Acyltransferases for alteration of polyunsaturated fatty acids and oil content in oleaginous yeasts

ABSTRACT

Two acyltransferases are provided, suitable for use in the manufacture of microbial oils enriched in omega fatty acids in oleaginous yeast (e.g.,  Yarrowia lipolytica ). Specifically, the genes encoding phophatidylcholine-diacylglycerol acyltransferase (PDAT) and diacylglycerol acyltransferase (DGAT2) have been isolated from  Y. lipolytica.  These genes encode enzymes that participate in the terminal step in oil biosynthesis in yeast. Each is expected to play a key role in altering the quantity of polyunsaturated fatty acids produced in oils of oleaginous yeasts.

This application claims the benefit of U.S. Provisional Application No.60/484,599, filed Jul. 2, 2003.

FIELD OF THE INVENTION

This invention is in the field of biotechnology. More specifically, thisinvention pertains to the identification of nucleic acid fragmentsencoding phospholipid:diacylglycerol acyltransferase and diacylglycerolacyltransferase. These enzymes are useful for altering the quantity ofoil in oleaginous microorganisms, such as oleaginous yeasts.

BACKGROUND OF THE INVENTION

The present invention is directed toward the development of anoleaginous yeast that accumulates oils enriched in long-chain ω-3 and/orω-6 polyunsaturated fatty acids (“PUFAs”; e.g., 18:3, 18:4, 20:3, 20:4,20:5, 22:6 fatty acids). Thus, in addition to developing techniques tointroduce the appropriate fatty acid desaturases and elongases intothese particular host organisms (where naturally produced PUFAs areusually limited to production of 18:2 fatty acids [and less commonly,18:3 fatty acids]), it is also necessary to increase the transfer ofPUFAs into storage lipid pools following their synthesis.

Most free fatty acids become esterified to coenzyme A (CoA), to yieldacyl-CoAs. These molecules are then substrates for glycerolipidsynthesis in the endoplasmic reticulum of the cell, where phosphatidicacid and diacylglycerol (DAG) are produced. Either of these metabolicintermediates may be directed to membrane phospholipids (e.g.,phosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine) orDAG may be directed to form triacylglycerols (TAGs), the primary storagereserve of lipids in eukaryotic cells.

In the yeast Saccharomyces cerevisiae, three pathways have beendescribed for the synthesis of TAGs. First, TAGs are mainly synthesizedfrom DAG and acyl-CoAs by the activity of diacylglycerolacyltransferases. More recently, however, a phospholipid:diacylglycerolacyltransferase has also been identified that is responsible forconversion of phospholipid and DAG to lysophospholipid and TAG,respectively, thus producing TAG via an acyl-CoA-independent mechanism(Dahlqvist et al., PNAS. 97(12):6487-6492 (2000)). Finally, twoacyl-CoA:sterol-acyltransferases are known that utilize acyl-CoAs andsterols to produce sterol esters (and TAGs in low quantities; seeSandager et al., Biochem. Soc. Trans. 28(6):700-702 (2000)).

A comprehensive mini-review on TAG biosynthesis in yeast, includingdetails concerning the genes involved and the metabolic intermediatesthat lead to TAG synthesis, is that of D. Sorger and G. Daum (Appl.Microbiol. Biotechnol. 61:289-299 (2003)). However, the authorsacknowledge that most work performed thus far has focused onSaccharomyces cerevisiae and numerous questions regarding TAG formationand regulation remain. In this organism it has been conclusivelydemonstrated that only four genes are involved in storage lipidsynthesis: ARE1 and ARE2 (encoding acyl-CoA:sterol-acyltransferases),LRO1 (encoding a phospholipid:diacylglycerol acyltransferase, or PDATenzyme) and DGA1 (encoding a diacylglycerol acyltransferase, or DGAT2enzyme) (Sandager, L. et al., J. Biol. Chem. 277(8):6478-6482 (2002)).Homologs of these genes have been identified in various other organismsand disclosed in the public literature, but none of these genes havebeen isolated from oleaginous yeast. Furthermore, techniques formodifying the transfer of fatty acids to the TAG pool in oleaginousyeast have not been developed. Thus, there is a need for theidentification and isolation of genes encoding acyltransferases thatwill be suitable for use in the production and accumulation of PUFAs inthe storage lipid pools (i.e., TAG fraction) of oleaginous yeast.

Genera typically identified as oleaginous yeast include, but are notlimited to: Yarrowia, Candida, Rhodotorula, Rhodosporidium,Cryptococcus, Trichosporon and Lipomyces. More specifically,illustrative oleaginous yeasts include: Rhodosporidium toruloides,Lipomyces starkeyii, L. lipoferus, Candida revkaufi, C. pulcherrima, C.tropicalis, C. utilis, Trichosporon pullans, T. cutaneum, Rhodotorulaglutinus, R. graminis and Yarrowia lipolytica (formerly classified asCandida lipolytica). These organisms can accumulate oil up to 80% oftheir dry cell weight; and, the technology for growing oleaginous yeastwith high oil content is well developed (for example, see EP 0 005277B1; Ratledge, C., Prog. Ind. Microbiol. 16:119-206 (1982)). Mostrecently, the natural abilities of oleaginous yeast (mostly limited to18:2 fatty acid production) have been enhanced by advances in geneticengineering, leading to the production of 20:4 (arachidonic acid), 20:5(eicosapentaenoic acid) and 22:6 (docosahexaenoic acid) PUFAs intransformant Yarrowia lipolytica. These ω-3 and ω-6 fatty acids wereproduced by introducing and expressing heterologous genes encoding theω-3/ω-6 biosynthetic pathway in the oleaginous host (see co-pending U.S.application Ser. No. 10/840,579).

The importance of PUFAs are undisputed. For example, certain PUFAs areimportant biological components of healthy cells and are recognized as:“essential” fatty acids that cannot be synthesized de novo in mammalsand instead must be obtained either in the diet or derived by furtherdesaturation and elongation of linoleic acid (LA) or α-linolenic acid(ALA); constituents of plasma membranes of cells, where they may befound in such forms as phospholipids or TAGs; necessary for properdevelopment (particularly in the developing infant brain) and for tissueformation and repair; and, precursors to several biologically activeeicosanoids of importance in mammals (e.g., prostacyclins, eicosanoids,leukotrienes, prostaglandins). Additionally, a high intake of long-chainω-3 PUFAs produces cardiovascular protective effects (Dyerberg, J. etal., Amer. J. Clin Nutr. 28:958-966 (1975); Dyerberg, J. et al., Lancet2(8081):117-119 (Jul. 15, 1978); Shimokawa, H., World Rev Nutr Diet,88:100-108 (2001); von Schacky, C., and Dyerberg, J., World Rev NutrDiet, 88:90-99 (2001)). And, numerous other studies documentwide-ranging health benefits conferred by administration of ω-3 and/orω-6 fatty acids against a variety of symptoms and diseases (e.g.,asthma, psoriasis, eczema, diabetes, cancer).

PUFAs are generally divided into two major classes (consisting of theω-6 and the ω-3 fatty acids) that are derived by desaturation andelongation of the essential fatty acids, LA and ALA, respectively.Despite a variety of commercial sources of PUFAs from natural sources[e.g., seeds of evening primrose, borage and black currants; filamentousfungi (Mortierella), Porphyridium (red alga), fish oils and marineplankton (Cyclotella, Nitzschia, Crypthecodinium)], there are severaldisadvantages associated with these methods of production (e.g., highlyheterogeneous oil compositions, accumulation of environmentalpollutants, uncontrollable fluctuations in availability due toweather/disease, expense at the commercial scale). As a result of theselimitations, extensive work has been conducted toward: 1.) thedevelopment of recombinant sources of PUFAs that are easy to producecommercially; and 2.) modification of fatty acid biosynthetic pathways,to enable production of desired PUFAs. Advances in the isolation,cloning and manipulation of fatty acid desaturase and elongase genesfrom various organisms have been made over the last several years.Knowledge of these gene sequences offers the prospect of producing adesired fatty acid and/or fatty acid composition in novel host organismsthat do not naturally produce PUFAs.

As described in Picataggio et al. (co-pending U.S. patent applicationSer. No. 10/840,579), oleaginous yeast have been identified as anappropriate microbial system in which to express PUFA desaturase andelongase genes to enable economical production of commercial quantitiesof one or more PUFAs in these particular hosts. To further advance thework described therein towards the development of an oleaginous yeastthat accumulates oils enriched in ω-3 and/or ω-6 fatty acids, however,it is necessary to increase the transfer of these PUFAs into storageTAGs (oil), once they are synthesized by fatty acid desaturases andelongases. Thus, there is a need for the identification and isolation ofgenes encoding acyltransferases that will be suitable for use in theproduction and accumulation of PUFAs in TAGs. Techniques for modifyingthe transfer of fatty acids to the TAG pool in oleaginous yeasts mustalso be developed.

Applicants have solved the stated problem by isolating the genesencoding PDAT and DGAT2 from the oleaginous yeast, Yarrowia lipolytica.These genes will be useful to enable one to modify the transfer of freefatty acids (e.g., ω-3 and/or ω-6 fatty acids) to the TAG pool inoleaginous yeast.

SUMMARY OF THE INVENTION

The invention relates to the discover of two genes, one encoding aphospholipid:diacylglycerol acyltransferase enzyme and the otherencoding a diacylglycerol acyltransferase enzyme, from Yarrowia. Thegenes and encoded enzymes are useful in manipulating the production ofcommercially useful oils in microorganisms, and particularly inoleaginous yeasts. Accordingly the invention provides an isolatednucleic acid molecule encoding an diacylglycerol acyltransferase enzyme,selected from the group consisting of:

-   -   (a) an isolated nucleic acid molecule encoding the amino acid        sequence selected from the group consisting of SEQ ID NOs:31, 78        and 79;    -   (b) an isolated nucleic acid molecule that hybridizes with (a)        under the following hybridization conditions: 0.1×SSC, 0.1% SDS,        65° C. and washed with 2×SSC, 0.1% SDS followed by 0.1×SSC, 0.1%        SDS; or    -   (c) an isolated nucleic acid molecule that is completely        complementary to (a) or (b).

In another embodiment the invention provides an isolated nucleic acidmolecule encoding an phospholipid:diacylglycerol acyltransferase enzyme,selected from the group consisting of:

-   -   (a) an isolated nucleic acid molecule encoding the amino acid        sequence as set forth in SEQ ID NO:46;    -   (b) an isolated nucleic acid molecule that hybridizes with (a)        under the following hybridization conditions: 0.1×SSC, 0.1% SDS,        65° C. and washed with 2×SSC, 0.1% SDS followed by 0.1×SSC, 0.1%        SDS; or    -   (c) an isolated nucleic acid molecule that is completely        complementary to (a) or (b).

Similarly the invention provides polypeptides having diacylglycerolacyltransferase and phospholipid:diacylglycerol acyltransferase activityencoded by the isolated nucleic acid molecules of the invention as wellas genetic chimera of these molecules and host cells comprising thesame.

In one preferred embodiment the invention provides a method ofincreasing triacylglycerol content in a transformed host cellcomprising:

-   (a) providing a transformed host cell comprising:    -   (i) at least one gene encoding an acyltransferase enzyme having        the amino acid sequence selected from the group consisting of        SEQ ID NOs:31, 78, 79 and 46 under the control of suitable        regulatory sequences; and    -   (ii) a source of fatty acids;-   (b) growing the cell of step (a) under conditions whereby the at    least one gene encoding an acyltransferase enzyme is expressed,    resulting in the transfer of the fatty acids to triacylglycerol; and-   (c) optionally recovering the triacylglycerol of step (b).

In an additional preferred embodiment the invention provides a method ofincreasing the ω-3 or ω-6 fatty acid content of triacylglycerols in atransformed host cell comprising:

-   (a) providing a transformed host cell comprising:    -   (i) at least one gene encoding at least one enzyme of the        ω-3/ω-6 fatty acid biosynthetic pathway;    -   (ii) at least one gene encoding an acyltransferase enzyme having        the amino acid sequence selected from the group consisting of        SEQ ID NOs:31, 78, 79 and 46 under the control of suitable        regulatory sequences;-   (b) growing the cell of step (a) under conditions whereby the genes    of (i) and (ii) are expressed, resulting in the production of at    least one ω-3 or ω-6 fatty acid and its transfer to triacylglycerol;    and-   (c) optionally recovering the triacylglycerol of step (b).

BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCE DESCRIPTIONS

FIG. 1 shows a schematic illustration of the biochemical mechanism forlipid accumulation in oleaginous yeast.

FIG. 2 illustrates the ω-3 and ω-6 fatty acid biosynthetic pathways.

FIG. 3 illustrates the construction of the plasmid vectors pY5 andpY5-13 for gene expression in Yarrowia lipolytica.

FIG. 4A shows a pairwise comparison between various yeast and fungalDGAT2 enzymes using a Clustal W analysis. In contrast, FIG. 4B shows apairwise comparison between various yeast and fungal PDAT enzymes.

FIGS. 5A and 5B show an alignment of known glyceraldehyde-3-phosphatedehydrogenase (GPD) proteins from Saccharomyces cerevisiae (GenBankAccession No. CAA24607), Schizosaccharomyces pombe (GenBank AccessionNo. NP_(—)595236), Aspergillus oryzae (GenBank Accession No. AAK08065),Paralichthys olivaceus (GenBank Accession No. BAA88638), Xenopus laevis(GenBank Accession No. P51469) and Gallus gallus (GenBank Accession No.DECHG3), used to identify two conserved regions within the sequencealignment.

The invention can be more fully understood from the following detaileddescription and the accompanying sequence descriptions, which form apart of this application.

The following sequences comply with 37 C.F.R. §1.821-1.825(“Requirements for Patent Applications Containing Nucleotide Sequencesand/or Amino Acid Sequence Disclosures—the Sequence Rules”) and areconsistent with World Intellectual Property Organization (WIPO) StandardST.25 (1998) and the sequence listing requirements of the EPO and PCT(Rules 5.2 and 49.5(a-bis), and Section 208 and Annex C of theAdministrative Instructions). The symbols and format used for nucleotideand amino acid sequence data comply with the rules set forth in 37C.F.R. §1.822.

SEQ ID NOs:1 and 2 correspond to primers TEF5′ and TEF3′, respectively,used to isolate the TEF promoter.

SEQ ID NOs:3 and 4 correspond to primers XPR5′ and XPR3′, respectively,used to isolate the XPR2 transcriptional terminator.

SEQ ID NOs:5-16 correspond to primers YL5, YL6, YL9, YL10, YL7, YL8,YL3, YL4, YL1, YL2, YL61 and YL62, respectively, used for plasmidconstruction.

SEQ ID NO:17 corresponds to a 1 kB DNA fragment (amino acid sequenceprovided as SEQ ID NO:18) containing the E. coli hygromycin resistancegene.

SEQ ID NO:19 corresponds to a 1.7 kB DNA fragment containing theYarrowia Ura3 gene (amino acid sequence provided as SEQ ID NO:20), whichwas amplified with primers KU5 and KU3 (SEQ ID NOs:21 and 22,respectively).

SEQ ID NOs:23 and 25 are the degenerate primers identified as P7 and P8,respectively, used for the isolation of a Yarrowia lipolytica DGAT2.

SEQ ID NOs:24 and 26 are the amino acid consensus sequences thatcorrespond to the degenerate primers P7 and P8, respectively.

SEQ ID NOs:27-29 correspond to primers P80, P81 and LinkAmp Primer1,respectively, used for chromosome walking.

SEQ ID NO:30 shows a 2119 bp DNA sequence comprising an ORF that encodesthe Y. lipolytica DGAT2. SEQ ID NO:31 is 514 amino acid residues inlength and corresponds to nucleotides +291 to +1835 of SEQ ID NO:30; SEQID NO:78 is 459 amino acid residues in length and corresponds tonucleotides +456 to +1835 of SEQ ID NO:30; and, SEQ ID NO:79 is 355amino acid residues in length and corresponds to nucleotides +768 to+1835 of SEQ ID NO:30, as set forth in SEQ ID NO:86.

SEQ ID NOs:32-35 correspond to primers P95, P96, P97 and P98,respectively, used for targeted disruption of the Y. lipolytica DGAT2gene.

SEQ ID NOs:36-38 correspond to primers P115, P116 and P112,respectively, used to screen for targeted integration of the disruptedY. lipolytica DGAT2 gene.

SEQ ID NOs:39 and 41 are the degenerate primers identified as P26 andP27, respectively, used for the isolation of the Y. lipolytica PDAT.

SEQ ID NOs:40 and 42 are the amino acid consensus sequences thatcorrespond to degenerate primers P26 and P27, respectively.

SEQ ID NOs:43 and 44 correspond to primers P39 and P42, respectively,used to amplify a 1008 bp portion of the Y. lipolytica PDAT gene.

SEQ ID NO:45 shows a DNA sequence that encodes the Y. lipolytica PDAT(ORF=nucleotides +274 to +2217), while SEQ ID NO:46 shows thecorresponding amino acid sequence of PDAT.

SEQ ID NOs:47 and 48 correspond to primers P41 and P40, respectively,used for targeted disruption of the Y. lipolytica PDAT gene.

SEQ ID NOs:49-52 correspond to primers P51, P52, P37 and P38,respectively, used to screen for targeted integration of the disruptedY. lipolytica PDAT gene.

SEQ ID NO:53 corresponds to primer P79, used to amplify the full-lengthY. lipolytica DGAT2 gene from rescued plasmids.

SEQ ID NOs:54 and 55 correspond to primers P84 and P85, respectively,used to amplify the full-length Y. lipolytica PDAT gene from rescuedplasmids.

SEQ ID NO:56 corresponds to a 971 bp fragment designated as “GPDPro”,and identified as the putative glyceraldehyde-3-phosphate dehydrogenase(GPD) promoter in Y. lipolytica.

SEQ ID NOs:57-62 correspond to the GPD amino acid sequences ofSaccharomyces cerevisiae (GenBank Accession No. CAA24607),Schizosaccharomyces pombe (GenBank Accession No. NP_(—)595236),Aspergillus oryzae (GenBank Accession No. AAK08065), Paralichthysolivaceus (GenBank Accession No. BAA88638), Xenopus laevis (GenBankAccession No. P51469) and Gallus gallus (GenBank Accession No. DECHG3),respectively.

SEQ ID NOs:63 and 64 correspond to conserved amino acid regions of theGPD protein.

SEQ ID NOs:65 and 66 correspond to the degenerate primers YL193 andYL194, respectively, used for isolating an internal portion of the Y.lipolytica GPD gene.

SEQ ID NO:67 encodes a 507 bp internal portion of the Y. lipolytica GPDgene, while SEQ ID NO:68 is the corresponding amino acid sequence.

SEQ ID NOs:69-71 correspond to primers YL206, YL207 and YL208,respectively, used for chromosome walking.

SEQ ID NO:72 corresponds to a 1848 bp fragment designated as “GPDP”,comprising 1525 bp upstream of the GPD gene and an additional 323 bprepresenting a 5′ portion of the GPD gene in Y. lipolytica.

SEQ ID NOs:73 and 74 correspond to primers P145 and P146, respectively,used to amplify the full-length Y. lipolytica DGAT2 gene.

SEQ ID NOs:75 and 76 correspond to primers YPDAT5 and YPDAT3,respectively, used to amplify the full-length Y. lipolytica PDAT gene.

SEQ ID NO:77 corresponds to primer LinkAmp primer 2, used for chromosomewalking.

SEQ ID NOs:80 and 81 correspond to primers GPD-1 and GPD-2,respectively, used to amplify the S. cerevisiaeglyceraldehyde-3-phosphate dehydrogenase (GPD) promoter.

SEQ ID NOs:82 and 83 correspond to primers ADHT-1 and ADHT-2,respectively, used to amplify the S. cerevisiae alcohol dehydrogenase(ADH1) terminator.

SEQ ID NOs:84 and 85 correspond to primers UP 161 and LP 162,respectively, used to create a S. cerevisiae LRO 1 targeting cassette.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the subject invention, Applicants have isolated andconfirmed the identity of Yarrowia lipolytica genes encodingphospholipid:diacylglycerol acyltransferase (PDAT) and diacylglycerolacyltransferase (DGAT2) enzymes useful for transferring fatty acids intostorage triacylglycerols (TAGs). This may be useful to alter thequantity of long chain polyunsaturated fatty acids (PUFAs) produced inoleaginous yeasts.

The subject invention finds many applications. PUFAs, or derivativesthereof, accumulated by the methodology disclosed herein can be used asdietary substitutes, or supplements, particularly infant formulas, forpatients undergoing intravenous feeding or for preventing or treatingmalnutrition. Alternatively, the purified PUFAs (or derivatives thereof)may be incorporated into cooking oils, fats or margarines formulated sothat in normal use the recipient would receive the desired amount fordietary supplementation. The PUFAs may also be incorporated into infantformulas, nutritional supplements or other food products and may finduse as anti-inflammatory or cholesterol lowering agents. Optionally, thecompositions may be used for pharmaceutical use (human or veterinary).In this case, the PUFAs are generally administered orally but can beadministered by any route by which they may be successfully absorbed,e.g., parenterally (e.g., subcutaneously, intramuscularly orintravenously), rectally, vaginally or topically (e.g., as a skinointment or lotion).

Supplementation of humans or animals with PUFAs produced by recombinantmeans can result in increased levels of the added PUFAs, as well astheir metabolic progeny. For example, treatment with arachidonic (ARA)can result not only in increased levels of ARA, but also downstreamproducts of ARA such as prostaglandins. Complex regulatory mechanismscan make it desirable to combine various PUFAs, or add differentconjugates of PUFAs, in order to prevent, control or overcome suchmechanisms to achieve the desired levels of specific PUFAs in anindividual.

Definitions

In this disclosure, a number of terms and abbreviations are used. Thefollowing definitions are provided.

“Open reading frame” is abbreviated ORF.

“Polymerase chain reaction” is abbreviated PCR.

“American Type Culture Collection” is abbreviated ATCC.

“Polyunsaturated fatty acid(s)” is abbreviated PUFA(s).

“Phospholipid:diacylglycerol acyltransferase” is abbreviated PDAT.

“Diacylglycerol acyltransferase” is abbreviated DGAT.

“Diacylglycerol” is abbreviated DAG.

“Triacylglycerols” are abbreviated TAGs.

“Co-enzyme A” is abbreviated CoA.

The term “fatty acids” refers to long chain aliphatic acids (alkanoicacids) of varying chain length, from about C₁₂ to C₂₂ (although bothlonger and shorter chain-length acids are known). The predominant chainlengths are between C₁₆ and C₂₂. The structure of a fatty acid isrepresented by a simple notation system of “X:Y”, where X is the totalnumber of carbon (C) atoms in the particular fatty acid and Y is thenumber of double bonds.

Generally, fatty acids are classified as saturated or unsaturated. Theterm “saturated fatty acids” refers to those fatty acids that have no“double bonds” between their carbon backbone. In contrast, “unsaturatedfatty acids” have “double bonds” along their carbon backbones (which aremost commonly in the cis-configuration). “Monounsaturated fatty acids”have only one “double bond” along the carbon backbone (e.g., usuallybetween the 9^(th) and 10^(th) carbon atom as for palmitoleic acid(16:1) and oleic acid (18:1)), while “polyunsaturated fatty acids” (or“PUFAs”) have at least two double bonds along the carbon backbone (e.g.,between the 9^(th) and 10^(th), and 12^(th) and 13^(th) carbon atoms forlinoleic acid (18:2); and between the 9^(th) and 10^(th), 12^(th) and13^(th), and 15^(th) and 16^(th) for α-linolenic acid (18:3)).

“PUFAs” can be classified into two major families (depending on theposition (n) of the first double bond nearest the methyl end of thefatty acid carbon chain). Thus, the “omega-6 fatty acids” (ω-6 or n-6)have the first unsaturated double bond six carbon atoms from the omega(methyl) end of the molecule and additionally have a total of two ormore double bonds, with each subsequent unsaturation occurring 3additional carbon atoms toward the carboxyl end of the molecule. Incontrast, the “omega-3 fatty acids” (ω-3 or n-3) have the firstunsaturated double bond three carbon atoms away from the omega end ofthe molecule and additionally have a total of three or more doublebonds, with each subsequent unsaturation occuring 3 additional carbonatoms toward the carboxyl end of the molecule.

For the purposes of the present disclosure, the omega-reference systemwill be used to indicate the number of carbons, the number of doublebonds and the position of the double bond closest to the omega carbon,counting from the omega carbon (which is numbered 1 for this purpose).This nomenclature is shown below in Table 1, in the column titled“Shorthand Notation”. The remainder of the Table summarizes the commonnames of ω-3 and ω-6 fatty acids, the abbreviations that will be usedthroughout the specification and each compounds' chemical name. TABLE 1Nomenclature Of Polyunsaturated Fatty Acids Shorthand Common NameAbbreviation Chemical Name Notation Linoleic LA cis-9,12-octadecadienoic18:2 ω-6 γ-Linoleic GLA cis-6,9,12- 18:3 ω-6 octadecatrienoic Dihomo-γ-DGLA cis-8,11,14- 20:3 ω-6 Linoleic eicosatrienoic Arachidonic ARAcis-5,8,11,14- 20:4 ω-6 eicosatetraenoic α-Linolenic ALA cis-9,12,15-18:3 ω-3 octadecatrienoic Stearidonic STA cis-6,9,12,15- 18:4 ω-3octadecatetraenoic Eicosatetraenoic ETA cis-8,11,14,17- 20:4 ω-3eicosatetraenoic Eicosapentaenoic EPA cis-5,8,11,14,17- 20:5 ω-3eicosapentaenoic Docosapentaenoic DPA cis-7,10,13,16,19- 22:5 ω-3docosapentaenoic Docosahexaenoic DHA cis-4,7,10,13,16,19- 22:6 ω-3docosahexaenoic

“Microbial oils” or “single cell oils” are those oils naturally producedby microorganisms (e.g., algae, oleaginous yeasts and filamentous fungi)during their lifespan. The term “oil” refers to a lipid substance thatis liquid at 25° C. and usually polyunsaturated. In contrast, the term“fat” refers to a lipid substance that is solid at 25° C. and usuallysaturated.

“Lipid bodies” refer to lipid droplets that usually are bounded byspecific proteins and a monolayer of phospholipid. These organelles aresites where most organisms transport/store neutral lipids. Lipid bodiesare thought to arise from microdomains of the endoplasmic reticulum thatcontain TAG-biosynthesis enzymes; and, their synthesis and size appearto be controlled by specific protein components.

“Neutral lipids” refer to those lipids commonly found in cells in lipidbodies as storage fats and oils and are so called because at cellularpH, the lipids bear no charged groups. Generally, they are completelynon-polar with no affinity for water. Neutral lipids generally refer tomono-, di-, and/or triesters of glycerol with fatty acids, also calledmonoacylglycerol, diacylglycerol or TAG, respectively (or collectively,acylglycerols). A hydolysis reaction must occur to release free fattyacids from acylglycerols.

The terms “triacylglycerol”, “oil” and “TAGs” refer to neutral lipidscomposed of three fatty acyl residues esterified to a glycerol molecule(and such terms will be used interchangeably throughout the presentdisclosure herein). Such oils can contain long chain PUFAs, as well asshorter saturated and unsaturated fatty acids and longer chain saturatedfatty acids. Thus, “oil biosynthesis” generically refers to thesynthesis of TAGs in the cell.

The term “DAG AT” refers to a diacylglycerol acyltransferase (also knownas an acyl-CoA-diacylglycerol acyltransferase or a diacylglycerolO-acyltransferase) (EC 2.3.1.20). This enzyme is responsible for theconversion of acyl-CoA and 1,2-diacylglycerol to TAG and CoA (therebyinvolved in the terminal step of TAG biosynthesis). Two families of DAGAT enzymes exist: DGAT1 and DGAT2. The former family shares homologywith the acyl-CoA:cholesterol acyltransferase (ACAT) gene family, whilethe latter family is unrelated (Lardizabal et al., J. Biol. Chem.276(42):38862-28869 (2001)). A representative DGAT2 enzyme is encoded bythe DGA1 gene of Saccharomyces cerevisiae (locus NP_(—)014888 of GenbankAccession No. NC_(—)001147; Oelkers et. al. J. Biol. Chem. 277:8877(2002)); a gene encoding DGAT2 isolated from Yarrowia lipolytica isprovided as SEQ ID NO:30.

The term “PDAT” refers to a phospholipid:diacylglycerol acyltransferaseenzyme (EC 2.3.1.158). This enzyme is responsible for the transfer of anacyl group from the sn-2 position of a phospholipid to the sn-3 positionof 1,2-diacylglycerol, thus resulting in lysophospholipid and TAG(thereby involved in the terminal step of TAG biosynthesis). This enzymediffers from DGAT (EC 2.3.1.20) by synthesizing TAG via anacyl-CoA-independent mechanism. A representative PDAT enzyme is encodedby the LRO1 gene in Saccharomyces cerevisiae (Dahlqvist et al., Proc.Natl. Acad. Sci. USA 97:6487 (2000)); a gene encoding PDAT isolated fromYarrowia lipolytica is provided as SEQ ID NO:45.

The term “PUFA biosynthetic pathway enzyme” refers to any of thefollowing enzymes (and genes which encode said enzymes) associated withthe biosynthesis of a PUFA including: a Δ4 desaturase, a Δ5 desaturase,a Δ6 desaturase, a Δ12 desaturase, a Δ15 desaturase, a Δ17 desaturase, aΔ9 desaturase, a Δ8 desaturase and/or an elongase.

The term “ω-3/ω-6 fatty biosynthetic pathway” refers to genes encodingthe enzymatic pathway as illustrated in FIG. 2, providing for theconversion of oleic acid through various intermediates to DHA.

The term “desaturase” refers to a polypeptide that can desaturate, i.e.,introduce a double bond, in one or more fatty acids to produce a mono-or polyunsaturated fatty acid. Despite use of the omega-reference systemthroughout the specification in reference to specific fatty acids, it ismore convenient to indicate the activity of a desaturase by countingfrom the carboxyl end of the substrate using the delta-system. Ofparticular interest herein are: Δ12 desaturases that desaturate a fattyacid between the 12^(th) and 13^(th) carbon atoms numbered from thecarboxyl-terminal end of the molecule and that catalyze the conversionof oleic acid to LA; Δ15 desaturases that catalyze the conversion of LAto ALA; Δ17 desaturases that catalyze the conversion of ARA to EPAand/or DGLA to ETA; Δ6 desaturases that catalyze the conversion of LA toGLA and/or ALA to STA; Δ5 desaturases that catalyze the conversion ofDGLA to ARA and/or ETA to EPA; Δ4 desaturases that catalyze theconversion of DPA to DHA; Δ8 desaturases that catalyze the conversion ofeicosadienoic acid (EDA; C20:2) to DGLA and/or eicosatrienoic acid(ETrA; C20:3) to ETA; and Δ9 desaturases that catalyze the conversion ofpalmitate to palmitoleic acid (16:1) and/or stearate to oleic acid(18:1).

The term “elongase” refers to a polypeptide that can elongate a fattyacid carbon chain to produce an acid that is 2 carbons longer than thefatty acid substrate that the elongase acts upon. This process ofelongation occurs in a multi-step mechanism in association with fattyacid synthase, whereby CoA is the acyl carrier (Lassner et al., ThePlant Cell 8:281-292 (1996)). Briefly, malonyl-CoA is condensed with along-chain acyl-CoA to yield CO₂ and a β-ketoacyl-CoA (where the acylmoiety has been elongated by two carbon atoms). Subsequent reactionsinclude reduction to β-hydroxyacyl-CoA, dehydration to an enoyl-CoA anda second reduction to yield the elongated acyl-CoA. Examples ofreactions catalyzed by elongases are the conversion of GLA to DGLA, STAto ETA, and EPA to DPA. Accordingly, elongases can have differentspecificities. For example, a C_(16/18) elongase will prefer a C₁₆substrate, a C_(18/20) elongase will prefer a C₁₈ substrate and aC_(20/22) elongase will prefer a C₂₀ substrate. In like manner, a Δ9elongase is able to catalyze the conversion of LA and ALA toeicosadienoic acid (EDA; C20:2) and eicosatrienoic acid (ETrA; C20:3),respectively.

The terms “conversion efficiency” and “percent substrate conversion”refer to the efficiency by which a particular enzyme (e.g., a desaturaseor elongase) can convert substrate to product. The conversion efficiencyis measured according.to the following formula:([product]/[substrate+product])*100, where ‘product’ includes theimmediate product and all products in the pathway derived from it.

The term “oleaginous” refers to those organisms that have the ability tostore their energy source in the form of TAGs (Weete, In: Fungal LipidBiochemistry, 2^(nd) ed., Plenum, 1980). Generally, the cellular oilcontent of these microorganisms follows a sigmoid curve, wherein theconcentration of lipid increases until it reaches a maximum at the latelogarithmic or early stationary growth phase and then graduallydecreases during the late stationary and death phases (Yongmanitchai andWard, Appl. Environ. Microbiol. 57:419-25 (1991)).

The term “oleaginous yeast” refers to those microorganisms classified asyeasts that can accumulate at least 25% of their dry cell weight as oil.Examples of oleaginous yeast include, but are no means limited to, thefollowing genera: Yarrowia, Candida, Rhodotorula, Rhodosporidium,Cryptococcus, Trichosporon and Lipomyces.

The term “fermentable carbon substrate” means a carbon source that amicroorganism will metabolize to derive energy. Typical carbonsubstrates of the invention include, but are not limited to:monosaccharides, oligosaccharides, polysaccharides, alkanes, fattyacids, esters of fatty acids, monoglycerides, carbon dioxide, methanol,formaldehyde, formate and carbon-containing amines.

As used herein, an “isolated nucleic acid fragment” is a polymer of RNAor DNA that is single- or double-stranded, optionally containingsynthetic, non-natural or altered nucleotide bases. An isolated nucleicacid fragment in the form of a polymer of DNA may be comprised of one ormore segments of cDNA, genomic DNA or synthetic DNA.

A nucleic acid molecule is “hybridizable” to another nucleic acidmolecule, such as a cDNA, genomic DNA, or RNA molecule, when asingle-stranded form of the nucleic acid molecule can anneal to theother nucleic acid molecule under the appropriate conditions oftemperature and solution ionic strength. Hybridization and washingconditions are well known and exemplified in Sambrook, J., Fritsch, E.F. and Maniatis, T. Molecular Cloning: A Laboratory Manual, 2^(nd) ed.,Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y. (1989),particularly Chapter 11 and Table 11.1 therein (entirely incorporatedherein by reference). The conditions of temperature and ionic strengthdetermine the “stringency” of the hybridization. Stringency conditionscan be adjusted to screen for moderately similar fragments (such ashomologous sequences from distantly related organisms), to highlysimilar fragments (such as genes that duplicate functional enzymes fromclosely related organisms). Post-hybridization washes determinestringency conditions. One set of preferred conditions uses a series ofwashes starting with 6×SSC, 0.5% SDS at room temperature for 15 min,then repeated with 2×SSC, 0.5% SDS at 45° C. for 30 min, and thenrepeated twice with 0.2×SSC, 0.5% SDS at 50° C. for 30 min. A morepreferred set of stringent conditions uses higher temperatures in whichthe washes are identical to those above except for the temperature ofthe final two 30 min washes in 0.2×SSC, 0.5% SDS was increased to 60° C.Another preferred set of highly stringent conditions uses two finalwashes in 0.1×SSC, 0.1% SDS at 65° C. An additional set of stringentconditions include hybridization at 0.1×SSC, 0.1% SDS, 65° C. and washedwith 2×SSC, 0.1% SDS followed by 0.1×SSC, 0.1% SDS, for example.

Hybridization requires that the two nucleic acids contain complementarysequences, although depending on the stringency of the hybridization,mismatches between bases are possible. The appropriate stringency forhybridizing nucleic acids depends on the length of the nucleic acids andthe degree of complementation, variables well known in the art. Thegreater the degree of similarity or homology between two nucleotidesequences, the greater the value of Tm for hybrids of nucleic acidshaving those sequences. The relative stability (corresponding to higherTm) of nucleic acid hybridizations decreases in the following order:RNA:RNA, DNA:RNA, DNA:DNA. For hybrids of greater than 100 nucleotidesin length, equations for calculating Tm have been derived (see Sambrooket al., supra, 9.50-9.51). For hybridizations with shorter nucleicacids, i.e., oligonucleotides, the position of mismatches becomes moreimportant, and the length of the oligonucleotide determines itsspecificity (see Sambrook et al., supra, 11.7-11.8). In one embodimentthe length for a hybridizable nucleic acid is at least about 10nucleotides. Preferably a minimum length for a hybridizable nucleic acidis at least about 15 nucleotides; more preferably at least about 20nucleotides; and most preferably the length is at least about 30nucleotides. Furthermore, the skilled artisan will recognize that thetemperature and wash solution salt concentration may be adjusted asnecessary according to factors such as length of the probe.

A “substantial portion” of an amino acid or nucleotide sequence is thatportion comprising enough of the amino acid sequence of a polypeptide orthe nucleotide sequence of a gene to putatively identify thatpolypeptide or gene, either by manual evaluation of the sequence by oneskilled in the art, or by computer-automated sequence comparison andidentification using algorithms such as BLAST (Basic Local AlignmentSearch Tool; Altschul, S. F., et al., J. Mol. Biol. 215:403-410 (1993).In general, a sequence of ten or more contiguous amino acids or thirtyor more nucleotides is necessary in order to putatively identify apolypeptide or nucleic acid sequence as homologous to a known protein orgene. Moreover, with respect to nucleotide sequences, gene specificoligonucleotide probes comprising 20-30 contiguous nucleotides may beused in sequence-dependent methods of gene identification (e.g.,Southern hybridization) and isolation (e.g., in situ hybridization ofbacterial colonies or bacteriophage plaques). In addition, shortoligonucleotides of 12-15 bases may be used as amplification primers inPCR in order to obtain a particular nucleic acid fragment comprising theprimers. Accordingly, a “substantial portion” of a nucleotide sequencecomprises enough of the sequence to specifically identify and/or isolatea nucleic acid fragment comprising the sequence. The instantspecification teaches partial or complete amino acid and nucleotidesequences encoding one or more particular yeast proteins. The skilledartisan, having the benefit of the sequences as reported herein, may nowuse all or a substantial portion of the disclosed sequences for purposesknown to those skilled in this art. Accordingly, the instant inventioncomprises the complete sequences as reported in the accompanyingSequence Listing, as well as substantial portions of those sequences asdefined above.

The term “complementary” is used to describe the relationship betweennucleotide bases that are capable of hybridizing to one another. Forexample, with respect to DNA, adenosine is complementary to thymine andcytosine is complementary to guanine. Accordingly, the instant inventionalso includes isolated nucleic acid fragments that are complementary tothe complete sequences as reported in the accompanying Sequence Listing,as well as those substantially similar nucleic acid sequences.

The term “percent identity”, as known in the art, is a relationshipbetween two or more polypeptide sequences or two or more polynucleotidesequences, as determined by comparing the sequences. In the art,“identity” also means the degree of sequence relatedness betweenpolypeptide or polynucleotide sequences, as the case may be, asdetermined by the match between strings of such sequences. “Identity”and “similarity” can be readily calculated by known methods, includingbut not limited to those described in: 1.) Computational MolecularBiology (Lesk, A. M., Ed.) Oxford University: NY (1988); 2.)Biocomputing: Informatics and Genome Proiects (Smith, D. W., Ed.)Academic: NY (1993); 3.) Computer Analysis of Sequence Data, Part I(Griffin, A. M., and Griffin, H. G., Eds.) Humania: NJ (1994); 4.)Sequence Analysis in Molecular Biology (von Heinje, G., Ed.) Academic(1987); and 5.) Sequence Analysis Primer (Gribskov, M. and Devereux, J.,Eds.) Stockton: NY (1991). Preferred methods to determine identity aredesigned to give the best match between the sequences tested. Methods todetermine identity and similarity are codified in publicly availablecomputer programs. Sequence alignments and percent identity calculationsmay be performed using the Megalign program of the LASERGENEbioinformatics computing suite (DNASTAR Inc., Madison, Wis.). Multiplealignment of the sequences is performed using the Clustal method ofalignment (Higgins and Sharp, CABIOS. 5:151-153 (1989)) with defaultparameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Default parametersfor pairwise alignments using the Clustal method are: KTUPLE 1, GAPPENALTY=3, WINDOW=5 and DIAGONALS SAVED=5.

Suitable nucleic acid fragments (isolated polynucleotides of the presentinvention) encode polypeptides that are at least about 70% identical,preferably at least about 75% identical, and more preferably at leastabout 80% identical to the amino acid sequences reported herein.Preferred nucleic acid fragments encode amino acid sequences that areabout 85% identical to the amino acid sequences reported herein. Morepreferred nucleic acid fragments encode amino acid sequences that are atleast about 90% identical to the amino acid sequences reported herein.Most preferred are nucleic acid fragments that encode amino acidsequences that are at least about 95% identical to the amino acidsequences reported herein. Suitable nucleic acid fragments not only havethe above homologies but typically encode a polypeptide having at least50 amino acids, preferably at least 100 amino acids, more preferably atleast 150 amino acids, still more preferably at least 200 amino acids,and most preferably at least 250 amino acids.

The term “sequence analysis software” refers to any computer algorithmor software program that is useful for the analysis of nucleotide oramino acid sequences. “Sequence analysis software” may be commerciallyavailable or independently developed. Typical sequence analysis softwarewill include, but is not limited to: 1.) the GCG suite of programs(Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison,Wis.); 2.) BLASTP, BLASTN, BLASTX (Altschul et al., J. Mol. Biol.215:403-410 (1990)); 3.) DNASTAR (DNASTAR, Inc. Madison, Wis.); 4.)Sequencher (Gene Codes Corporation, Ann Arbor, Mich.); and 5.) the FASTAprogram incorporating the Smith-Waterman algorithm (W. R. Pearson,Comput. Methods Genome Res., [Proc. Int. Symp.] (1994), Meeting Date1992, 111-20. Editor(s): Suhai, Sandor. Plenum: New York, N.Y.). Withinthe context of this application it will be understood that wheresequence analysis software is used for analysis, that the results of theanalysis will be based on the “default values” of the programreferenced, unless otherwise specified. As used herein “default values”will mean any set of values or parameters that originally load with thesoftware when first initialized.

“Codon degeneracy” refers to the nature in the genetic code permittingvariation of the nucleotide sequence without affecting the amino acidsequence of an encoded polypeptide. The skilled artisan is well aware ofthe “codon-bias” exhibited by a specific host cell in usage ofnucleotide codons to specify a given amino acid. Therefore, whensynthesizing a gene for improved expression in a host cell, it isdesirable to design the gene such that its frequency of codon usageapproaches the frequency of preferred codon usage of the host cell.

The term “codon-optimized”, as it refers to genes or coding regions ofnucleic acid molecules, refers to modification of codons such that thealtered codons reflect the typical codon usage of the host organismwithout altering the polypeptide for which the DNA codes.

“Synthetic genes” can be assembled from oligonucleotide building blocksthat are chemically synthesized using procedures known to those skilledin the art. These building blocks are ligated and annealed to form genesegments that are then enzymatically assembled to construct the entiregene. Accordingly, the genes can be tailored for optimal gene expressionbased on optimization of nucleotide sequence to reflect the codon biasof the host cell. The skilled artisan appreciates the likelihood ofsuccessful gene expression if codon usage is biased towards those codonsfavored by the host. Determination of preferred codons can be based on asurvey of genes derived from the host cell, where sequence informationis available.

“Gene” refers to a nucleic acid fragment that expresses a specificprotein, including regulatory sequences preceding (5′ non-codingsequences) and following (3′ non-coding sequences) the coding sequence.“Native gene” refers to a gene as found in nature with its ownregulatory sequences. “Chimeric gene” refers to any gene that is not anative gene, comprising regulatory and coding sequences that are notfound together in nature. Accordingly, a chimeric gene may compriseregulatory sequences and coding sequences that are derived fromdifferent sources, or regulatory sequences and coding sequences derivedfrom the same source, but arranged in a manner different than that foundin nature. “Endogenous gene” refers to a native gene in its naturallocation in the genome of an organism. A “foreign” gene refers to a genenot normally found in the host organism, but that is introduced into thehost organism by gene transfer. Foreign genes can comprise native genesinserted into a non-native organism, or chimeric genes. A “transgene” isa gene that has been introduced into the genome by a transformationprocedure. A “codon-optimized gene” is a gene having its frequency ofcodon usage designed to mimic the frequency of preferred codon usage ofthe host cell.

“Coding sequence” refers to a DNA sequence that codes for a specificamino acid sequence. “Suitable regulatory sequences” refer to nucleotidesequences located upstream (5′ non-coding sequences), within, ordownstream (3′ non-coding sequences) of a coding sequence, and whichinfluence the transcription, RNA processing or stability, or translationof the associated coding sequence. Regulatory sequences may includepromoters, translation leader sequences, introns, polyadenylationrecognition sequences, RNA processing sites, effector binding sites andstem-loop structures.

“Promoter” refers to a DNA sequence capable of controlling theexpression of a coding sequence or functional RNA. In general, a codingsequence is located 3′ to a promoter sequence. Promoters may be derivedin their entirety from a native gene, or be composed of differentelements derived from different promoters found in nature, or evencomprise synthetic DNA segments. It is understood by those skilled inthe art that different promoters may direct the expression of a gene indifferent tissues or cell types, or at different stages of development,or in response to different environmental or physiological conditions.Promoters that cause a gene to be expressed in most cell types at mosttimes are commonly referred to as “constitutive promoters”. It isfurther recognized that since in most cases the exact boundaries ofregulatory sequences have not been completely defined, DNA fragments ofdifferent lengths may have identical promoter activity.

The term “3′ non-coding sequences” or “transcription terminator” refersto DNA sequences located downstream of a coding sequence. This includespolyadenylation recognition sequences and other sequences encodingregulatory signals capable of affecting mRNA processing or geneexpression. The polyadenylation signal is usually characterized byaffecting the addition of polyadenylic acid tracts to the 3′ end of themRNA precursor. The 3′ region can influence the transcription, RNAprocessing or stability, or translation of the associated codingsequence.

“RNA transcript” refers to the product resulting from RNApolymerase-catalyzed transcription of a DNA sequence. When the RNAtranscript is a perfect complementary copy of the DNA sequence, it isreferred to as the primary transcript or it may be a RNA sequencederived from post-transcriptional processing of the primary transcriptand is referred to as the mature RNA. “Messenger RNA” or “mRNA” refersto the RNA that is without introns and that can be translated intoprotein by the cell. “cDNA” refers to a double-stranded DNA that iscomplementary to, and derived from, mRNA. “Sense” RNA refers to RNAtranscript that includes the mRNA and so can be translated into proteinby the cell. “Antisense RNA” refers to a RNA transcript that iscomplementary to all or part of a target primary transcript or mRNA andthat blocks the expression of a target gene (U.S. Pat. No. 5,107,065; WO99/28508). The complementarity of an antisense RNA may be with any partof the specific gene transcript, i.e., at the 5′ non-coding sequence, 3′non-coding sequence, or the coding sequence. “Functional RNA” refers toantisense RNA, ribozyme RNA, or other RNA that is not translated and yethas an effect on cellular processes.

The term “operably linked” refers to the association of nucleic acidsequences on a single nucleic acid fragment so that the function of oneis affected by the other. For example, a promoter is operably linkedwith a coding sequence when it is capable of affecting the expression ofthat coding sequence (i.e., the coding sequence is under thetranscriptional control of the promoter). Coding sequences can beoperably linked to regulatory sequences in sense or antisenseorientation.

The term “expression”, as used herein, refers to the transcription andstable accumulation of sense (mRNA) or antisense RNA derived from thenucleic acid fragment(s) of the invention. Expression may also refer totranslation of mRNA into a polypeptide.

“Transformation” refers to the transfer of a nucleic acid molecule intoa host organism, resulting in genetically stable inheritance. Thenucleic acid molecule may be a plasmid that replicates autonomously, forexample; or, it may integrate into the genome of the host organism. Hostorganisms containing the transformed nucleic acid fragments are referredto as “transgenic” or “recombinant” or “transformed” organisms.

The terms “plasmid”, “vector” and “cassette” refer to an extrachromosomal element often carrying genes that are not part of thecentral metabolism of the cell, and usually in the form of circulardouble-stranded DNA fragments. Such elements may be autonomouslyreplicating sequences, genome integrating sequences, phage or nucleotidesequences, linear or circular, of a single- or double-stranded DNA orRNA, derived from any source, in which a number of nucleotide sequenceshave been joined or recombined into a unique construction which iscapable of introducing a promoter fragment and DNA sequence for aselected gene product along with appropriate 3′ untranslated sequenceinto a cell. “Transformation cassette” refers to a specific vectorcontaining a foreign gene(s) and having elements in addition to theforeign gene(s) that facilitate transformation of a particular hostcell. “Expression cassette” refers to a specific vector containing aforeign gene(s) and having elements in addition to the foreign gene(s)that allow for enhanced expression of that gene in a foreign host.

The term “homologous recombination” refers to the exchange of DNAfragments between two DNA molecules (during cross over). The fragmentsthat are exchanged are flanked by sites of identical nucleotidesequences between the two DNA molecules (i.e., “regions of homology”).The term “regions of homology” refer to stretches of nucleotide sequenceon nucleic acid fragments that participate in homologous recombinationthat have homology to each other. Effective homologous recombinationwill take place where these regions of homology are at least about 10 bpin length where at least about 50 bp in length is preferred. Typicallyfragments that are intended for recombination contain at least tworegions of homology where targeted gene disruption or replacement isdesired.

Standard recombinant DNA and molecular cloning techniques used hereinare well known in the art and are described by Sambrook, J., Fritsch, E.F. and Maniatis, T., Molecular Cloning: A Laboratory Manual, 2^(nd) ed.,Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y. (1989)(hereinafter “Maniatis”); by Silhavy, T. J., Bennan, M. L. and Enquist,L. W., Experiments with Gene Fusions, Cold Spring Harbor Laboratory:Cold Spring Harbor, N.Y. (1984); and by Ausubel, F. M. et al., CurrentProtocols in Molecular Biology, published by Greene Publishing Assoc.and Wiley-lnterscience (1987).

Microbial Biosynthesis of Fatty Acids and Triacylglycerols

In general, lipid accumulation in oleaginous microorganisms is triggeredin response to the overall carbon to nitrogen ratio present in thegrowth medium (FIG. 1). When cells have exhausted available nitrogensupplies (e.g., when the carbon to nitrogen ratio is greater than about40), the depletion of cellular adenosine monophosphate (AMP) leads tothe cessation of AMP-dependent isocitrate dehydrogenase activity in themitochondria and the accumulation of citrate, transport of citrate intothe cytosol, and subsequent cleavage of the citrate by ATP-citrate lyaseto yield acetyl-CoA and oxaloacetate. Acetyl-CoA is the principlebuilding block for de novo biosynthesis of fatty acids. Although anycompound that can effectively be metabolized to acetyl-CoA can serve asa precursor of fatty acids, glucose is the primary source of carbon inthis type of reaction (FIG. 1). Glucose is converted to pyruvate viaglycolysis, and pyruvate is then transported into the mitochondria whereit can be converted to acetyl-CoA by pyruvate dehydrogenase (“PD”).Since acetyl-CoA can not be transported directly across themitochondrial membrane into the cytoplasm, the two carbons fromacetyl-CoA condense with oxaloacetate to yield citrate (catalyzed bycitrate synthase). Citrate is transported directly into the cytoplasm,where it is cleaved by ATP-citrate lyase to regenerate acetyl-CoA andoxaloacetate. The oxaloacetate reenters the tricarboxylic acid cycle,via conversion to malate.

The synthesis of malonyl-CoA is the first committed step of fatty acidbiosynthesis, which takes place in the cytoplasm. Malonyl-CoA isproduced via carboxylation of acetyl-CoA by acetyl-CoA carboxylase(“ACC”). Fatty acid synthesis is catalyzed by a multi-enzyme fatty acidsynthase complex (“FAS”) and occurs by the condensation of eighttwo-carbon fragments (acetyl groups from acetyl-CoA) to form a 16-carbonsaturated fatty acid, palmitate. More specifically, FAS catalyzes aseries of 7 reactions, which involve the following (Smith, S. FASEB J,8(15):1248-59 (1994)):

-   -   1. Acetyl-CoA and malonyl-CoA are transferred to the acyl        carrier peptide (ACP) of FAS. The acetyl group is then        transferred to the malonyl group, forming β-ketobutyryl-ACP and        releasing CO₂.    -   2. The β-ketobutyryl-ACP undergoes reduction (via β-ketoacyl        reductase) and dehydration (via β-hydroxyacyl dehydratase) to        form a trans-monounsaturated fatty acyl group.    -   3. The double bond is reduced by NADPH, yielding a saturated        fatty-acyl group two carbons longer than the initial one. The        butyryl-group's ability to condense with a new malonyl group and        repeat the elongation process is then regenerated.    -   4. When the fatty acyl group becomes 16 carbons long, a        thioesterase activity hydrolyses it, releasing free palmitate        (16:0).

Whereas palmitate synthesis occurs in the cytosol, formation of longerchain saturated and unsaturated fatty acid derivates occur in both themitochondria and endoplasmic reticulum (ER), wherein the ER is thedominant system. Specifically, palmitate (16:0) is the precursor ofstearic (18:0), palmitoleic (16:1) and oleic (18:1) acids through theaction of elongases and desaturases. For example, palmitate and stearateare converted to their unsaturated derivatives, palmitoleic (16:1) andoleic (18:1) acids, respectively, by the action of a Δ9 desaturase.

TAGs (the primary storage unit for fatty acids) are formed by a seriesof reactions that involve: 1.) the esterification of one molecule ofacyl-CoA to glycerol-3-phosphate via an acyltransferase to producelysophosphatidic acid; 2.) the esterification of a second molecule ofacyl-CoA via an acyltransferase to yield 1,2-diacylglycerol phosphate(commonly identified as phosphatidic acid); 3.) removal of a phosphateby phosphatidic acid phosphatase to yield 1,2-diacylglycerol (DAG); and4.) the addition of a third fatty acid by the action of a DAGacyltransferase (e.g., PDAT, DGAT2 or DGAT2) to form TAG (FIG. 1).

A wide spectrum of fatty acids can be incorporated into TAGs, includingsaturated and unsaturated fatty acids and short-chain and long-chainfatty acids. Some non-limiting examples of fatty acids that can beincorporated into TAGs by acyltransferases (e.g., DGAT2 or PDAT)include: capric (10:0), lauric (12:0), myristic (14:0), palmitic (16:0),palmitoleic (16:1), stearic (18:0), oleic (18:1), vaccenic (18:1),linoleic (18:2), eleostearic (18:3), γ-linolenic (18:3), α-linolenic(18:3), stearidonic (18:4), arachidic (20:0), eicosadienoic (20:2),dihomo-γ-linoleic (20:3), eicosatrienoic (20:3), arachidonic (20:4),eicosa-tetraenoic (20:4), eicosa-pentaenoic (20:5), behenic (22:0),docosa-pentaenoic (22:5), docosa-hexaenoic (22:6), lignoceric (24:0),nervonic (24:1), cerotic (26:0), and montanic (28:0) fatty acids. Inpreferred embodiments of the present invention, incorporation of PUFAsinto TAG is most desirable.

Genes Encoding DGAT2

Historically, DGAT1 (responsible for the third acyl transferasereaction, wherein an acyl-CoA group is transferred from acyl-CoA to thesn-3 position of DAG to form TAG) was thought to be the only enzymespecifically involved in TAG synthesis. This enzyme was known to behomologous to acyl-CoA:cholesterol acyltransferases (ACATs); however,recent studies have identified a new family of DAG acyltransferaseenzymes that are unrelated to the ACAT gene family. Thus, nomenclaturenow distinguishes between the DAG acyltransferase enzymes that arerelated to the ACAT gene family (DGAT1 family) versus those that areunrelated (DGAT2 family) (Lardizabal et al., J. Biol. Chem.276(42):38862-28869 (2001)). Members of the DGAT2 family appear to bepresent in all major phyla of eukaryotes (fungi, plants, animals, andbasal eukaryotes).

Many genes encoding DGAT2 enzymes have been identified through geneticmeans and the DNA sequences of some of these genes are publiclyavailable. For example, some non-limiting examples include the followingGenBank Accession Numbers: NC_(—)001147 (locus NP_(—)014888;Saccharomyces cerevisiae); NM_(—)012079 (human); NM_(—)127503, AF051849and AJ238008 (Arabidopsis thaliana); NM_(—)026384, NM_(—)010046 andAB057816 (mouse); AY093657 (pig); AB062762 (rat); AF221132(Caenorhabditis elegans); AF391089 and AF391090 (Mortierellaramanniana); AF129003 (Nicotiana tabacum); and, AF251794 and AF164434(Brassica napus). Additionally, the patent literature provides manyadditional DNA sequences of DGAT2 genes (and/or details concerningseveral of the genes above and their methods of isolation). See, forexample: US 2003/124126 (Cases et al.); US 2003/115632, US2003/0028923and US 2004/0107459 (Lardizabal et al.); and WO 2001/034814 (Banas etal.).

Despite disclosure of several complete and incomplete sequences encodingDGAT2 (supra), very few of these sequences have been shown to have DGAT2activity. The exceptions include the work of: 1.) Bouvier-Nave, P. etal. (Biochem. Soc. Trans. 28(6):692-695 (2000)), wherein the DGAT2 ofthe nematode worm Caenorhabditis elegans was expressed in Saccharomycescerevisiae, leading to an increase in TAG content and in microsomaloleyl-CoA:DAG acyltransferase activity; and, 2.) Lardizabal et al.(supra; see also US 2003/0028923 A1 and US 2004/0107459 A1), wherein twoDGAT2s of the fungus Mortierella ramanniana were expressed in insectcells, leading to high levels of DGAT activity on membranes isolatedfrom those cells. In addition to these demonstrations where oilbiosynthesis was increased by over-expression of DGAT2, disruption ofthe genes encoding DGAT2 have also been shown to result in a decrease inthe cellular TAG content (Oelkers et al. J Biol Chem. 277(11):8877-81(2002); Sandager et al., J Biol Chem. 277:6478-6482 (2002); Sorger andDaum. J. Bacteriol. 184:519-524 (2002)).

Genes Encoding PDAT

TAG synthesis can also occur in the absence of acyl-CoA, via theacyl-CoA-independent PDAT enzyme, as recently discovered by Dahlqvist etal. (Proc. Nat. Acad. Sci. (USA) 97:6487-6492 (2000)) and Oelkers et al.(J. Biol. Chem. 275:15609-15612 (2000)). Specifically, PDAT removes anacyl group from the sn-2 position of a phosphotidylcholine substrate fortransfer to the sn-3 position of DAG to produce TAG; and, although thefunction of PDAT is not as well characterized as DGAT2, PDAT has beenpostulated to play a major role in removing “unusual” fatty acids fromphospholipids in some oilseed plants (Banas, A. et al., Biochem. Soc.Trans. 28(6):703-705 (2000)).

PDAT is structurally related to the lecithin:cholesterol acyltransferase(LCAT) family of proteins. Several genes encoding PDAT enzymes have beenidentified through genetic means and the DNA sequences of some of thesegenes are publicly available. For example, some non-limiting examplesinclude the following GenBank Accession Numbers: P40345 (Saccharomycescerevisiae); O94680 and NP_(—)596330 (Schizosaccharomyces pombe); and,NP_(—)190069 and AB006704 [gi:2351069] Arabidopsis thaliana).Additionally, the patent literature provides many additional DNAsequences of PDAT genes (and/or details concerning several of the genesabove and their methods of isolation); see, for example, WO 2000/060095(Dahlqvist et al.).

In a manner similar to DGAT2, over-expression of PDAT has beenaccomplished in Saccharomyces cerevisiae to increase oil biosynthesis.For example, over-expressing the S. cerevisiae LRO1 gene encoding PDATresulted in an increased TAG content, confirming the involvement of thisenzyme in TAG formation (Dahlqvist et al. Proc. Nat. Acad. Sci. (USA)97:6487-6492 (2000); Oelkers et al., J. Biol. Chem. 275:15609-15612(2000)). In contrast, deletion of the LRO1 gene was found to causesignificant reduction of TAG synthesis (Oelkers et al., supra).

Biosynthesis of Omega-3 and Omega-6 Polyunsaturated Fatty Acids

The metabolic process that converts LA to GLA, DGLA and ARA (the ω-6pathway) and ALA to STA, ETA, EPA, DPA and DHA (the ω-3 pathway)involves elongation of the carbon chain through the addition oftwo-carbon units and desaturation of the molecule through the additionof double bonds (FIG. 2). This requires a series of desaturation andelongation enzymes. Specifically, oleic acid is converted to LA (18:2),the first of the ω-6 fatty acids, by the action of a Δ12 desaturase.Subsequent ω-6 fatty acids are produced as follows: 1.) LA is convertedto GLA by the activity of a Δ6 desaturase; 2.) GLA is converted to DGLAby the action of an elongase; and 3.) DGLA is converted to ARA by theaction of a Δ5 desaturase. In like manner, linoleic acid (LA) isconverted to ALA, the first of the ω-3 fatty acids, by the action of aΔ15 desaturase. Subsequent ω-3 fatty acids are produced in a series ofsteps similar to that for the ω-6 fatty acids. Specifically, 1.) ALA isconverted to STA by the activity of a Δ6 desaturase; 2.) STA isconverted to ETA by the activity of an elongase; and 3.) ETA isconverted to EPA by the activity of a Δ5 desaturase. Alternatively, ETAand EPA can be produced from DGLA and ARA, respectively, by the activityof a Δ17 desaturase. EPA can be further converted to DHA by the activityof an elongase and a Δ4 desaturase.

In alternate embodiments, a Δ9 elongase is able to catalyze theconversion of LA and ALA to eicosadienoic acid (EDA; C20:2) andeicosatrienoic acid (ETrA; C20:3), respectively. A Δ8 desaturase thenconverts these products to DGLA and ETA, respectively.

Many microorganisms, including algae, bacteria, molds, fungi and yeasts,can synthesize PUFAs and omega fatty acids in the ordinary course ofcellular metabolism. Particularly well-studied are fungi includingSchizochytrium aggregatm, species of the genus Thraustochytrium andMortierella alpina. Additionally, many dinoflagellates (Dinophyceaae) 15naturally produce high concentrations of PUFAs. As such, a variety ofdesaturase and elongase genes involved in PUFA production have beenidentified through genetic means and the DNA sequences of some of thesegenes are publicly available (non-limiting examples are shown below inTable 2): TABLE 2 Some Publicly Available Genes Involved In PUFAProduction Genbank Accession No. Description AY131238 Argania spinosa Δ6desaturase Y055118 Echium pitardii var. pitardii Δ6 desaturase AY055117Echium gentianoides Δ6 desaturase AF296076 Mucor rouxii Δ6 desaturaseAF007561 Borago officinalis Δ6 desaturase L11421 Synechocystis sp. Δ6desaturase NM_031344 Rattus norvegicus Δ6 fatty acid desaturaseAF465283, Mortierella alpina Δ6 fatty acid desaturase AF465281, AF110510AF465282 Mortierella isabellina Δ6 fatty acid desaturase AF419296Pythium irregulare Δ6 fatty acid desaturase AB052086 Mucorcircinelloides D6d mRNA for Δ6 fatty acid desaturase AJ250735 Ceratodonpurpureus mRNA for Δ6 fatty acid desaturase AF126799 Homo sapiens Δ6fatty acid desaturase AF126798 Mus musculus Δ6 fatty acid desaturaseAF199596, Homo sapiens Δ5 desaturase AF226273 AF320509 Rattus norvegicusliver Δ5 desaturase AB072976 Mus musculus D5D mRNA for Δ5 desaturaseAF489588 Thraustochytrium sp. ATCC21685 Δ5 fatty acid desaturaseAJ510244 Phytophthora megasperma mRNA for Δ5 fatty acid desaturaseAF419297 Pythium irregulare Δ5 fatty acid desaturase AF07879Caenorhabditis elegans Δ5 fatty acid desaturase AF067654 Mortierellaalpina Δ5 fatty acid desaturase AB022097 Dictyostelium discoideum mRNAfor Δ5 fatty acid desaturase AF489589.1 Thraustochytrium sp. ATCC21685Δ4 fatty acid desaturase AAG36933 Emericella nidulans oleate Δ12desaturase AF110509 Mortierella alpina Δ12 fatty acid desaturase mRNAAB020033 Mortierella alpina mRNA for Δ12 fatty acid desaturase AAL13300Mortierella alpina Δ12 fatty acid desaturase AF417244 Mortierella alpinaATCC 16266 Δ12 fatty acid desaturase gene AF161219 Mucor rouxii Δ12desaturase mRNA AY332747 Pavlova lutheri Δ4 fatty acid desaturase (des1)mRNA AAG36933 Emericella nidulans oleate Δ12 desaturase AF110509,Mortierella alpina Δ12 fatty acid desaturase mRNA AB020033 AAL13300Mortierella alpina Δ12 fatty acid desaturase AF417244 Mortierella alpinaATCC 16266 Δ12 fatty acid desaturase AF161219 Mucor rouxii Δ12desaturase mRNA X86736 Spiruline platensis Δ12 desaturase AF240777Caenorhabditis elegans Δ12 desaturase AB007640 Chlamydomonas reinhardtiiΔ12 desaturase AB075526 Chlorella vulgaris Δ12 desaturase AP002063Arabidopsis thaliana microsomal Δ12 desaturase NP_441622, Synechocystissp. PCC 6803 Δ15 desaturase BAA18302, BAA02924 AAL36934 Perillafrutescens Δ15 desaturase AF338466 Acheta domesticus Δ9 desaturase 3mRNA AF438199 Picea glauca desaturase Δ9 (Des9) mRNA E11368 Anabaena Δ9desaturase E11367 Synechocystis Δ9 desaturase D83185 Pichia angusta DNAfor Δ9 fatty acid desaturase U90417 Synechococcus vulcanus Δ9 acyl-lipidfatty acid desaturase (desC) gene AF085500 Mortierella alpina Δ9desaturase mRNA AY504633 Emericella nidulans Δ9 stearic acid desaturase(sdeB) gene NM_069854 Caenorhabditis elegans essential fatty aciddesaturase, stearoyl-CoA desaturase (39.1 kD) (fat-6) complete mRNAAF230693 Brassica oleracea cultivar Rapid Cycling stearoyl-ACPdesaturase (Δ9-BO-1) gene, exon sequence AX464731 Mortierella alpinaelongase gene (also WO 02/08401) NM_119617 Arabidopsis thaliana fattyacid elongase 1 (FAE1) (At4g34520) mRNA NM_134255 Mus musculus ELOVLfamily member 5, elongation of long chain fatty acids (yeast) (Elovl5),mRNA NM_134383 Rattus norvegicus fatty acid elongase 2 (rELO2), mRNANM_134382 Rattus norvegicus fatty acid elongase 1 (rELO1), mRNANM_068396, Caenorhabditis elegans fatty acid ELOngation (elo-6), (elo-NM_068392, 5), (elo-2), (elo-3), and (elo-9) mRNA NM_070713, NM_068746,NM_064685

Additionally, the patent literature provides many additional DNAsequences of genes (and/or details concerning several of the genes aboveand their methods of isolation) involved in PUFA production. See, forexample: U.S. Pat. No. 5,968,809 (Δ6 desaturases); U.S. Pat. No.5,972,664 and U.S. Pat. No. 6,075,183 (Δ5 desaturases); WO 91/13972 andU.S. Pat. No. 5,057,419 (Δ9 desaturases); WO 93/11245 (Δ15 desaturases);WO 94/11516, U.S. Pat. No. 5,443,974 and WO 03/099216 (Δ12 desaturases);WO 00/12720 and U.S. 2002/0139974A1 (elongases); U.S. 2003/0196217 A1(Δ17 desaturase); WO 00/34439 (Δ8 desaturases); and, WO 02/090493 (Δ4desaturases).

Each of these patents and applications are herein incorporated byreference in their entirety.

Depending upon the host cell, the availability of substrate, and thedesired end product(s), several desaturases and elongases are ofinterest for use in production of PUFAs. Considerations for choosing aspecific polypeptide having desaturase or elongase activity include: 1.)the substrate specificity of the polypeptide; 2.) whether thepolypeptide or a component thereof is a rate-limiting enzyme; 3.)whether the desaturase or elongase is essential for synthesis of adesired PUFA; and/or 4.) co-factors required by the polypeptide. Theexpressed polypeptide preferably has parameters compatible with thebiochemical environment of its location in the host cell. For example,the polypeptide may have to compete for substrate with other enzymes inthe host cell. Analyses of the K_(M) and specific activity of thepolypeptide are therefore considered in determining the suitability of agiven polypeptide for modifying PUFA production in a given host cell.The polypeptide used in a particular host cell is one that can functionunder the biochemical conditions present in the intended host cell butotherwise can be any polypeptide having desaturase or elongase activitycapable of modifying the desired fatty acid substrate.

Sequence Identification of Yarrowia lipolytica DGAT2 and PDATAcyltransferases

Despite the availability of several genes encoding DGAT2 and PDAT(supra) which could be used for heterologous expression in oleaginousyeast (e.g., Yarrowia lipolytica), expression of a native enzyme ispreferred over a heterologous (or “foreign”) enzyme whenever possible.This preference occurs because: 1.) the native enzyme is optimized forinteraction with other enzymes and proteins in the cell; and 2.)heterologous genes are unlikely to share the same codon preference inthe host organism. Knowledge of the sequences of a host organism'snative PDAT and DGAT2 genes also facilitates disruption of thehomologous chromosomal genes by targeted disruption. And, as the presentinvention has shown, disruption of one or more of an organism'sacyltransferases (e.g., PDAT, DGAT2), when at least one acyltransferaseremains functional, can result in altered oil content.

Comparison of the PDAT nucleotide base (SEQ ID NO:45) and deduced aminoacid (SEQ ID NO:46) sequences to some public databases reveals that themost similar known sequences are about 47.1% identical to the amino acidsequence of PDAT reported herein over a length of 648 amino acids usingthe Clustal W method of alignment (Higgins and Sharp, CABIOS. 5:151-153(1989)). More preferred amino acid fragments are at least about 70%-80%identical to the sequences herein, where those sequences that are85%-90% identical are particularly suitable and those sequences that areabout 95% identical are most preferred. Similarly, preferred PDATencoding nucleic acid sequences corresponding to the instant ORF arethose encoding active proteins and which are at least about 70%-80%identical to the nucleic acid sequences encoding PDAT reported herein,where those sequences that are 85%-90% identical are particularlysuitable and those sequences that are about 95% identical are mostpreferred.

Comparison of the DGAT2 nucleotide base (SEQ ID NO:30) and deduced aminoacid (SEQ ID NO:79) sequences to some public databases reveals that themost similar known sequences are about 38.4% identical to the amino acidsequence of DGAT2 reported herein over a length of 355 amino acids usingthe Clustal W method of alignment (Higgins and Sharp, supra). Morepreferred amino acid fragments are at least about 70%-80% identical tothe sequences herein, where those sequences that are 85%-90% identicalare particularly suitable and those sequences that are about 95%identical are most preferred. Similarly, preferred DGAT2 encodingnucleic acid sequences corresponding to the instant ORF are thoseencoding active proteins and which are at least about 70%-80% identicalto the nucleic acid sequences encoding DGAT2 reported herein, wherethose sequences that are 85%-90% identical are particularly suitable andthose sequences that are about 95% identical are most preferred.

Isolation of Homologs

Each of the acyltransferase nucleic acid fragments of the instantinvention may be used to isolate genes encoding homologous proteins fromthe same or other microbial species. Isolation of homologous genes usingsequence-dependent protocols is well known in the art. Examples ofsequence-dependent protocols include, but are not limited to: 1.)methods of nucleic acid hybridization; 2.) methods of DNA and RNAamplification, as exemplified by various uses of nucleic acidamplification technologies [e.g., polymerase chain reaction (PCR),Mullis et al., U.S. Pat. No. 4,683,202; ligase chain reaction (LCR),Tabor, S. et al., Proc. Acad. Sci. USA 82:1074 (1985); or stranddisplacement amplification (SDA), Walker, et al., Proc. Natl. Acad. Sci.U.S.A., 89:392 (1992)]; and 3.) methods of library construction andscreening by complementation.

For example, genes encoding similar proteins or polypeptides to theacyltransferases described herein could be isolated directly by usingall or a portion of the instant nucleic acid fragments as DNAhybridization probes to screen libraries from any desired yeast orfungus using methodology well known to those skilled in the art.Specific oligonucleotide probes based upon the instant nucleic acidsequences can be designed and synthesized by methods known in the art(Maniatis, supra). Moreover, the entire sequences can be used directlyto synthesize DNA probes by methods known to the skilled artisan (e.g.,random primers DNA labeling, nick translation or end-labelingtechniques), or RNA probes using available in vitro transcriptionsystems. In addition, specific primers can be designed and used toamplify a part of (or full-length of) the instant sequences. Theresulting amplification products can be labeled directly duringamplification reactions or labeled after amplification reactions, andused as probes to isolate full-length DNA fragments under conditions ofappropriate stringency.

Typically, in PCR-type amplification techniques, the primers havedifferent sequences and are not complementary to each other. Dependingon the desired test conditions, the sequences of the primers should bedesigned to provide for both efficient and faithful replication of thetarget nucleic acid. Methods of PCR primer design are common and wellknown in the art (Thein and Wallace, “The use of oligonucleotides asspecific hybridization probes in the Diagnosis of Genetic Disorders”, inHuman Genetic Diseases: A Practical Approach, K. E. Davis Ed., (1986) pp33-50, IRL: Herndon, Va.; and Rychlik, W., In Methods in MolecularBiology, White, B. A. Ed., (1993) Vol. 15, pp 31-39, PCR Protocols:Current Methods and Applications. Humania: Totowa, N.J.).

Generally two short segments of the instant sequences may be used inpolymerase chain reaction protocols to amplify longer nucleic acidfragments encoding homologous genes from DNA or RNA. The polymerasechain reaction may also be performed on a library of cloned nucleic acidfragments wherein the sequence of one primer is derived from the instantnucleic acid fragments, and the sequence of the other primer takesadvantage of the presence of the polyadenylic acid tracts to the 3′ endof the mRNA precursor encoding microbial genes.

Alternatively, the second primer sequence may be based upon sequencesderived from the cloning vector. For example, the skilled artisan canfollow the RACE protocol (Frohman et al., PNAS USA 85:8998 (1988)) togenerate cDNAs by using PCR to amplify copies of the region between asingle point in the transcript and the 3′ or 5′ end. Primers oriented inthe 3′ and 5′ directions can be designed from the instant sequences.Using commercially available 3′ RACE or 5′ RACE systems (BRL,Gaithersburg, Md.), specific 3′ or 5′ cDNA fragments can be isolated(Ohara et al., PNAS USA 86:5673 (1989); Loh et al., Science 243:217(1989)).

Alternatively, the instant acyltransferase sequences may be employed ashybridization reagents for the identification of homologs. The basiccomponents of a nucleic acid hybridization test include a probe, asample suspected of containing the gene or gene fragment of interest,and a specific hybridization method. Probes of the present invention aretypically single-stranded nucleic acid sequences that are complementaryto the nucleic acid sequences to be detected. Probes are “hybridizable”to the nucleic acid sequence to be detected. The probe length can varyfrom 5 bases to tens of thousands of bases, and will depend upon thespecific test to be done. Typically a probe length of about 15 bases toabout 30 bases is suitable. Only part of the probe molecule need becomplementary to the nucleic acid sequence to be detected. In addition,the complementarity between the probe and the target sequence need notbe perfect. Hybridization does occur between imperfectly complementarymolecules with the result that a certain fraction of the bases in thehybridized region are not paired with the proper complementary base.

Hybridization methods are well defined. Typically the probe and samplemust be mixed under conditions that will permit nucleic acidhybridization. This involves contacting the probe and sample in thepresence of an inorganic or organic salt under the proper concentrationand temperature conditions. The probe and sample nucleic acids must bein contact for a long enough time that any possible hybridizationbetween the probe and sample nucleic acid may occur. The concentrationof probe or target in the mixture will determine the time necessary forhybridization to occur. The higher the probe or target concentration,the shorter the hybridization incubation time needed. Optionally, achaotropic agent may be added. The chaotropic agent stabilizes nucleicacids by inhibiting nuclease activity. Furthermore, the chaotropic agentallows sensitive and stringent hybridization of short oligonucleotideprobes at room temperature (Van Ness and Chen, Nucl. Acids Res.19:5143-5151 (1991)). Suitable chaotropic agents include guanidiniumchloride, guanidinium thiocyanate, sodium thiocyanate, lithiumtetrachloroacetate, sodium perchlorate, rubidium tetrachloroacetate,potassium iodide and cesium trifluoroacetate, among others. Typically,the chaotropic agent will be present at a final concentration of about 3M. If desired, one can add formamide to the hybridization mixture,typically 30-50% (v/v).

Various hybridization solutions can be employed. Typically, thesecomprise from about 20 to 60% volume, preferably 30%, of a polar organicsolvent. A common hybridization solution employs about 30-50% v/vformamide, about 0.15 to 1 M sodium chloride, about 0.05 to 0.1 Mbuffers (e.g., sodium citrate, Tris-HCI, PIPES or HEPES (pH range about6-9)), about 0.05 to 0.2% detergent (e.g., sodium dodecylsulfate), orbetween 0.5-20 mM EDTA, FICOLL (Pharmacia Inc.) (about 300-500 kdal),polyvinylpyrrolidone (about 250-500 kdal) and serum albumin. Alsoincluded in the typical hybridization solution will be unlabeled carriernucleic acids from about 0.1 to 5 mg/mL, fragmented nucleic DNA (e.g.,calf thymus or salmon sperm DNA, or yeast RNA), and optionally fromabout 0.5 to 2% wt/vol glycine. Other additives may also be included,such as volume exclusion agents that include a variety of polarwater-soluble or swellable agents (e.g., polyethylene glycol), anionicpolymers (e.g., polyacrylate or polymethylacrylate) and anionicsaccharidic polymers (e.g., dextran sulfate).

Nucleic acid hybridization is adaptable to a variety of assay formats.One of the most suitable is the sandwich assay format. The sandwichassay is particularly adaptable to hybridization under non-denaturingconditions. A primary component of a sandwich-type assay is a solidsupport. The solid support has adsorbed to it or covalently coupled toit immobilized nucleic acid probe that is unlabeled and complementary toone portion of the sequence.

Availability of the instant nucleotide and deduced amino acid sequencesfacilitates immunological screening of DNA expression libraries.Synthetic peptides representing portions of the instant amino acidsequences may be synthesized. These peptides can be used to immunizeanimals to produce polyclonal or monoclonal antibodies with specificityfor peptides or proteins comprising the amino acid sequences. Theseantibodies can then be used to screen DNA expression libraries toisolate full-length DNA clones of interest (Lerner, R. A. Adv. Immunol.36:1 (1984); Maniatis, supra).

Gene Optimization for Improved Heterologous Expression

It may be desirable to modify the expression of particularacyltransferases and/or PUFA biosynthetic pathway enzymes to achieveoptimal conversion efficiency of each, according to the specific TAGcomposition of interest. As such, a variety of techniques can beutilized to improve/optimize the expression of a polypeptide of interestin an alternative host. Two such techniques include codon-optimizationand mutagenesis of the gene.

Codon Optimization

For the purposes of the present invention, it may be desirable to modifya portion of the codons encoding polypeptides having acyltransferaseactivity, for example, to enhance the expression of genes encoding thosepolypeptides in an alternate host (i.e., an oleaginous yeast other thanYarrowia lipolytica). In general, host-preferred codons can bedetermined within a particular host species of interest by examiningcodon usage in proteins (preferably those expressed in the largestamount) and determining which codons are used with highest frequency.Thus, the coding sequence for a polypeptide having acyltransferaseactivity can be synthesized in whole or in part using the codonspreferred in the host species. All (or portions) of the DNA also can besynthesized to remove any destabilizing sequences or regions ofsecondary structure that would be present in the transcribed mRNA. All(or portions) of the DNA also can be synthesized to alter the basecomposition to one more preferable in the desired host cell.

Mutagenesis

Methods for synthesizing sequences and bringing sequences together arewell established in the literature. For example, in vitro mutagenesisand selection, site-directed mutagenesis, error prone PCR (Melnikov etal., Nucleic Acids Research, 27(4):1056-1062 (Feb. 15, 1999)), “geneshuffling” or other means can be employed to obtain mutations ofnaturally occurring acyltransferase genes. This would permit productionof a polypeptide having acyltransferase activity in vivo with moredesirable physical and kinetic parameters for function in the host cell(e.g., a longer half-life or a higher rate of synthesis of TAGs fromfatty acids).

If desired, the regions of an acyltransferase polypeptide important forenzymatic activity can be determined through routine mutagenesis,expression of the resulting mutant polypeptides and determination oftheir activities. Mutants may include deletions, insertions and pointmutations, or combinations thereof. A typical functional analysis beginswith deletion mutagenesis to determine the N- and C-terminal limits ofthe protein necessary for function, and then internal deletions,insertions or point mutants are made to further determine regionsnecessary for function. Other techniques such as cassette mutagenesis ortotal synthesis also can be used. Deletion mutagenesis is accomplished,for example, by using exonucleases to sequentially remove the 5′ or 3′coding regions. Kits are available for such techniques. After deletion,the coding region is completed by ligating oligonucleotides containingstart or stop codons to the deleted coding region after the 5′ or 3′deletion, respectively. Alternatively, oligonucleotides encoding startor stop codons are inserted into the coding region by a variety ofmethods including site-directed mutagenesis, mutagenic PCR or byligation onto DNA digested at existing restriction sites. Internaldeletions can similarly be made through a variety of methods includingthe use of existing restriction sites in the DNA, by use of mutagenicprimers via site-directed mutagenesis or mutagenic PCR. Insertions aremade through methods such as linker-scanning mutagenesis, site-directedmutagenesis or mutagenic PCR. Point mutations are made throughtechniques such as site-directed mutagenesis or mutagenic PCR.

Chemical mutagenesis also can be used for identifying regions of anacyltransferase polypeptide important for activity. A mutated constructis expressed, and the ability of the resulting altered protein tofunction as an acyltransferase is assayed. Such structure-functionanalysis can determine which regions may be deleted, which regionstolerate insertions, and which point mutations allow the mutant proteinto function in substantially the same way as the native acyltransferase.

All such mutant proteins and nucleotide sequences encoding them that arederived from the acyltransferase genes described herein are within thescope of the present invention.

Microbial Production of Fatty Acids and Triacylglycerols

Microbial production of fatty acids and TAGs has several advantages overpurification from natural sources such as fish or plants. For example:

-   -   1.) Many microbes are known with greatly simplified oil        compositions compared with those of higher organisms, making        purification of desired components easier;    -   2.) Microbial production is not subject to fluctuations caused        by external variables, such as weather and food supply;    -   3.) Microbially produced oil is substantially free of        contamination by environmental pollutants; and,    -   4.) Microbial oil production can be manipulated by controlling        culture conditions, notably by providing particular substrates        for microbially expressed enzymes, or by addition of compounds        or genetic engineering approaches to suppress undesired        biochemical pathways.        With respect to the production of ω-3 and/or ω-6 fatty acids in        particular, and TAGs containing those PUFAs, additional        advantages are incurred since microbes can provide fatty acids        in particular forms that may have specific uses; and,        recombinant microbes provide the ability to alter the naturally        occurring microbial fatty acid profile by providing new        biosynthetic pathways in the host or by suppressing undesired        pathways, thereby increasing levels of desired PUFAs, or        conjugated forms thereof, and decreasing levels of undesired        PUFAs.

Thus, knowledge of the sequences of the present acyltransferase geneswill be useful for manipulating fatty acid biosynthesis and accumulationin oleaginous yeasts, and particularly, in Yarrowia lipolytica. This mayrequire metabolic engineering directly within the fatty acid or TAGbiosynthetic pathways or additional manipulation of pathways thatcontribute carbon to the fatty acid biosynthetic pathway. Methods usefulfor manipulating biochemical pathways are well known to those skilled inthe art.

Metabolic Engineering to Up-Regulate Genes and Biosynthetic PathwaysAffecting Fatty Acid Synthesis and Oil Accumulation in Oleaginous Yeast

It is expected that introduction of chimeric genes encoding theacyltransferases described herein, under the control of the appropriatepromoters, will result in increased transfer of fatty acids to storageTAGs. As such, the present invention encompasses a method for increasingthe TAG content in an oleaginous yeast comprising expressing at leastone acyltransferase enzyme of the present invention in a transformedoleaginous yeast host cell producing a fatty acid, such that the fattyacid is transferred to the TAG pool.

Additional copies of acyltransferase genes may be introduced into thehost to increase the transfer of fatty acids to the TAG fraction.Expression of the genes also can be increased at the transcriptionallevel through the use of a stronger promoter (either regulated orconstitutive) to cause increased expression, by removing/deletingdestabilizing sequences from either the mRNA or the encoded protein, orby adding stabilizing sequences to the mRNA (U.S. Pat. No. 4,910,141).Yet another approach to increase expression of heterologous genes is toincrease the translational efficiency of the encoded mRNAs byreplacement of codons in the native gene with those for optimal geneexpression in the selected host microorganism.

In one specific embodiment, the present invention encompasses a methodof increasing the ω-3 and/or ω-6 fatty acid content of TAGs in anoleaginous yeast, since it is possible to introduce an expressioncassette encoding each of the enzymes necessary for ω-3 and/or ω-6 fattyacid biosynthesis into the organism (since naturally produced PUFAs inthese organisms are limited to 18:2 (i.e., LA), and less commonly 18:3(i.e., ALA) fatty acids). Thus, the method comprises:

-   -   a) providing a transformed oleaginous yeast host cell        (possessing at least one gene encoding at least one enzyme of        the ω-3/ω-6 fatty acid biosynthetic pathway and at least one        acyltransferase enzyme of the present invention);    -   b) growing the yeast cells of step (a) in the presence of a        fermentable carbon substrate, whereby the gene(s) of the ω-3/ω-6        fatty acid biosynthetic pathway and the acyltransferase(s) are        expressed, whereby a ω-3 and/or ω-6 fatty acid is produced, and        whereby the ω-3 and/or ω-6 fatty acid is transferred to TAGs.

A variety of PUFA products can be produced (prior to their transfer toTAGs), depending on the fatty acid substrate and the particular genes ofthe ω-3/ω-6 fatty acid biosynthetic pathway that are transformed intothe host cell. As such, production of the desired fatty acid product canoccur directly (wherein the fatty acid substrate is converted directlyinto the desired fatty acid product without any intermediate steps orpathway intermediates) or indirectly (wherein multiple genes encodingthe PUFA biosynthetic pathway may be used in combination, such that aseries of reactions occur to produce a desired PUFA). Specifically, forexample, it may be desirable to transform an oleaginous yeast with anexpression cassette comprising a Δ12 desaturase, Δ6 desaturase, ahigh-affinity elongase, a Δ5 desaturase and a Δ17 desaturase for theoverproduction of EPA. As is well known to one skilled in the art,various other combinations of the following enzymatic activities may beuseful to express in a host in conjunction with the acyltransferasesdescribed herein: a Δ15 desaturase, a Δ4 desaturase, a Δ5 desaturase, aΔ6 desaturase, a Δ17 desaturase, a Δ9 desaturase, a Δ8 desaturase and/oran elongase (see FIG. 2). The particular genes included within aparticular expression cassette will depend on the host cell (and itsPUFA profile and/or desaturase profile), the availability of substrateand the desired end product(s).

Thus, within the context of the present invention, it may be useful tomodulate the expression of the TAG biosynthetic pathway by any one ofthe methods described above. For example, the present invention providesgenes encoding key enzymes in the fatty acid biosynthetic pathwayleading to the storage of TAGs. These genes encode the PDAT and DGAT2enzymes. It will be particularly useful to modify the expression levelsof these genes in oleaginous yeasts to maximize production andaccumulation of TAGs using various means for metabolic engineering ofthe host organism. In preferred embodiments, modification of theexpression levels for these genes in combination with expression ofω-3/ω-6 biosynthetic genes can be utilized to maximize production andaccumulation of preferred PUFAs in the TAG pool.

Metabolic Engineering to Down-Regulate Undesirable Genes andBiosynthetic Pathways Affecting Fatty Acid Synthesis and OilAccumulation in Oleaginous Yeast

In some embodiments, it may be useful to disrupt or inactivate a hostorganism's native acyltransferase(s), based on the complete sequencesdescribed herein, the complement of those complete sequences,substantial portions of those sequences, codon-optimized desaturasesderived therefrom, and those sequences that are substantially homologousthereto. For example, the targeted disruption of the DGAT2acyltransferase, PDAT acyltransferase, and DGAT2 and PDATacyltransferases (as a double knockout) described herein in Yarrowialipolytica produced mutant strains that each had different reducedlevels of oil production (Example 5).

For gene disruption, a foreign DNA fragment (typically a selectablemarker gene) is inserted into the structural gene to be disrupted inorder to interrupt its coding sequence and thereby functionallyinactivate the gene. Transformation of the disruption cassette into thehost cell results in replacement of the functional native gene byhomologous recombination with the non-functional disrupted gene (see,for example: Hamilton et al., J. Bacteriol. 171:4617-4622 (1989); Balbaset al., Gene 136:211-213 (1993); Gueldener et al., Nucleic Acids Res.24:2519-2524 (1996); and Smith et al., Methods Mol. Cell. Biol.5:270-277(1996)).

Antisense technology is another method of down-regulating genes when thesequence of the target gene is known. To accomplish this, a nucleic acidsegment from the desired gene is cloned and operably linked to apromoter such that the anti-sense strand of RNA will be transcribed.This construct is then introduced into the host cell and the antisensestrand of RNA is produced. Antisense RNA inhibits gene expression bypreventing the accumulation of mRNA that encodes the protein ofinterest. The person skilled in the art will know that specialconsiderations are associated with the use of antisense technologies inorder to reduce expression of particular genes. For example, the properlevel of expression of antisense genes may require the use of differentchimeric genes utilizing different regulatory elements known to theskilled artisan.

Although targeted gene disruption and antisense technology offereffective means of down-regulating genes where the sequence is known,other less specific methodologies have been developed that are notsequence-based. For example, cells may be exposed to UV radiation andthen screened for the desired phenotype. Mutagenesis with chemicalagents is also effective for generating mutants and commonly usedsubstances include chemicals that affect nonreplicating DNA (e.g., HNO₂and NH₂OH), as well as agents that affect replicating DNA (e.g.,acridine dyes, notable for causing frameshift mutations). Specificmethods for creating mutants using radiation or chemical agents are welldocumented in the art. See, for example: Thomas D. Brock inBiotechnology: A Textbook of Industrial Microbiology, 2^(nd) ed. (1989)Sinauer Associates: Sunderland, Mass.; or Deshpande, Mukund V., Appl.Biochem. Biotechnol., 36:227 (1992).

Another non-specific method of gene disruption is the use oftransposable elements or transposons. Transposons are genetic elementsthat insert randomly into DNA but can be later retrieved on the basis ofsequence to determine where the insertion has occurred. Both in vivo andin vitro transposition methods are known. Both methods involve the useof a transposable element in combination with a transposase enzyme. Whenthe transposable element or transposon is contacted with a nucleic acidfragment in the presence of the transposase, the transposable elementwill randomly insert into the nucleic acid fragment. The technique isuseful for random mutagenesis and for gene isolation, since thedisrupted gene may be identified on the basis of the sequence of thetransposable element. Kits for in vitro transposition are commerciallyavailable [see, for example: 1.) The Primer Island Transposition Kit,available from Perkin Elmer Applied Biosystems, Branchburg, N.J., basedupon the yeast Ty1 element; 2.) The Genome Priming System, availablefrom New England Biolabs, Beverly, Mass., based upon the bacterialtransposon Tn7; and 3.) the EZ::TN Transposon Insertion Systems,available from Epicentre Technologies, Madison, Wis., based upon the Tn5bacterial transposable element].

Thus, within the context of the present invention, it may be useful todisrupt one of the acyltransferase genes of the invention. For example,it may be necessary to disrupt genes and pathways that diminish theexisting fatty acid pool and/or that hydrolyze TAGs to regulate (and/ormaximize) TAG accumulation.

Expression Systems, Cassettes and Vectors

The genes and gene products of the instant sequences described hereinmay be produced in microbial host cells, particularly in the cells ofoleaginous yeasts (e.g., Yarrowia lipolytica). Expression in recombinantmicrobial hosts may be useful for the transfer of various fatty acids toTAGs.

Microbial expression systems and expression vectors containingregulatory sequences that direct high level expression of foreignproteins are well known to those skilled in the art. Any of these couldbe used to construct chimeric genes for production of any of the geneproducts of the instant sequences. These chimeric genes could then beintroduced into appropriate microorganisms via transformation to providehigh level expression of the encoded enzymes.

Vectors or DNA cassettes useful for the transformation of suitable hostcells are well known in the art. The specific choice of sequencespresent in the construct is dependent upon the desired expressionproducts (supra), the nature of the host cell and the proposed means ofseparating transformed cells versus non-transformed cells. Typically,however, the vector or cassette contains sequences directingtranscription and translation of the relevant gene(s), a selectablemarker and sequences allowing autonomous replication or chromosomalintegration. Suitable vectors comprise a region 5′ of the gene thatcontrols transcriptional initiation and a region 3′ of the DNA fragmentthat controls transcriptional termination. It is most preferred whenboth control regions are derived from genes from the transformed hostcell, although it is to be understood that such control regions need notbe derived from the genes native to the specific species chosen as aproduction host.

Initiation control regions or promoters which are useful to driveexpression of the instant ORFs in the desired host cell are numerous andfamiliar to those skilled in the art. Virtually any promoter capable ofdirecting expression of these genes in the selected host cell issuitable for the present invention. Expression in a host cell can beaccomplished in a transient or stable fashion. Transient expression canbe accomplished by inducing the activity of a regulatable promoteroperably linked to the gene of interest. Stable expression can beachieved by the use of a constitutive promoter operably linked to thegene of interest. As an example, when the host cell is yeast,transcriptional and translational regions functional in yeast cells areprovided, particularly from the host species. The transcriptionalinitiation regulatory regions can be obtained, for example, from: 1.)genes in the glycolytic pathway, such as alcohol dehydrogenase,glyceraldehyde-3-phosphate-dehydrogenase (see U.S. Patent ApplicationNo. 60/482,263, incorporated herein by reference), phosphoglyceratemutase (see U.S. Patent Application No. 60/482,263, incorporated hereinby reference), fructose-bisphosphate aldolase (see U.S. PatentApplication No. 60/519,971, incorporated herein by reference),phosphoglucose-isomerase, phosphoglycerate kinase, etc.; or, 2.)regulatable genes such as acid phosphatase, lactase, metallothionein,glucoamylase, the translation elongation factor EF1-α (TEF) protein(U.S. Pat. No. 6,265,185), ribosomal protein S7 (U.S. Pat. No.6,265,185), etc. Any one of a number of regulatory sequences can beused, depending upon whether constitutive or induced transcription isdesired, the efficiency of the promoter in expressing the ORF ofinterest, the ease of construction and the like.

Nucleotide sequences surrounding the translational initiation codon‘ATG’ have been found to affect expression in yeast cells. If thedesired polypeptide is poorly expressed in yeast, the nucleotidesequences of exogenous genes can be modified to include an efficientyeast translation initiation sequence to obtain optimal gene expression.For expression in yeast, this can be done by site-directed mutagenesisof an inefficiently expressed gene by fusing it in-frame to anendogenous yeast gene, preferably a highly expressed gene.Alternatively, one can determine the consensus translation initiationsequence in the host and engineer this sequence into heterologous genesfor their optimal expression in the host of interest.

The termination region can be derived from the 3′ region of the genefrom which the initiation region was obtained or from a different gene.A large number of termination regions are known and functionsatisfactorily in a variety of hosts (when utilized both in the same anddifferent genera and species from where they were derived). Thetermination region usually is selected more as a matter of conveniencerather than because of any particular property. Preferably, thetermination region is derived from a yeast gene, particularlySaccharomyces, Schizosaccharomyces, Candida, Yarrowia or Kluyveromyces.The 3′-regions of mammalian genes encoding γ-interferon and α-2interferon are also known to function in yeast. Termination controlregions may also be derived from various genes native to the preferredhosts. Optionally, a termination site may be unnecessary; however, it ismost preferred if included.

As one of skill in the art is aware, merely inserting a gene into acloning vector does not ensure that it will be successfully expressed atthe level needed. In response to the need for a high expression rate,many specialized expression vectors have been created by manipulating anumber of different genetic elements that control aspects oftranscription, translation, protein stability, oxygen limitation andsecretion from the host cell. More specifically, some of the molecularfeatures that have been manipulated to control gene expression include:1.) the nature of the relevant transcriptional promoter and terminatorsequences; 2.) the number of copies of the cloned gene and whether thegene is plasmid-borne or integrated into the genome of the host cell;3.) the final cellular location of the synthesized foreign protein; 4.)the efficiency of translation in the host organism; 5.) the intrinsicstability of the cloned gene protein within the host cell; and 6.) thecodon usage within the cloned gene, such that its frequency approachesthe frequency of preferred codon usage of the host cell. Each of thesetypes of modifications are encompassed in the present invention, asmeans to further optimize expression of the acyltransferase enzymes.

Preferred Microbial Hosts for Recombinant Expression of Acyltransferases

Host cells for expression of the instant genes and nucleic acidfragments may include microbial hosts that grow on a variety offeedstocks, including simple or complex carbohydrates, organic acids andalcohols and/or hydrocarbons over a wide range of temperature and pHvalues. Although the genes described in the instant invention have beenisolated for expression in an oleaginous yeast, and in particularYarrowia lipolytica, it is contemplated that because transcription,translation and the protein biosynthetic apparatus is highly conserved,any bacteria, yeast, algae and/or filamentous fungus will be a suitablehost for expression of the present nucleic acid fragments.

Preferred microbial hosts are oleaginous organisms, such as oleaginousyeasts. These oleaginous organisms are naturally capable of oilsynthesis and accumulation, wherein the total oil content can comprisegreater than about 25% of the cellular dry weight, more preferablygreater than about 30% of the cellular dry weight and most preferablygreater than about 40% of the cellular dry weight. Additionally, thereis basis for the use of these organisms for the production of PFUA's asseen in co-pending U.S. application Ser. No. 10/840,579, hereinincorporated entirely by reference.

Genera typically identified as oleaginous yeast include, but are notlimited to: Yarrowia, Candida, Rhodotorula, Rhodosporidium,Cryptococcus, Trichosporon and Lipomyces. More specifically,illustrative oil-synthesizing yeasts include: Rhodosporidium toruloides,Lipomyces starkeyii, L. lipoferus, Candida revkaufi, C. pulcherrima, C.tropicalis, C. utilis, Trichosporon pullans, T. cutaneum, Rhodotorulaglutinus, R. graminis, and Yarrowia lipolytica (formerly classified asCandida lipolytica).

Most preferred is the oleaginous yeast Yarrowia lipolytica; and, in afurther embodiment, most preferred are the Y. lipolytica strainsdesignated as ATCC #20362, ATCC #8862, ATCC #18944, ATCC #76982, ATCC#90812 and/or LGAM S(7)1 (Papanikolaou S., and Aggelis G., Bioresour.Technol. 82(1):43-9 (2002)).

Transformation of Microbial Hosts

Once the DNA encoding a polypeptide suitable for expression in anoleaginous yeast has been obtained, it is placed in a plasmid vectorcapable of autonomous replication in a host cell or it is directlyintegrated into the genome of the host cell. Integration of expressioncassettes can occur randomly within the host genome or can be targetedthrough the use of constructs containing regions of homology with thehost genome sufficient to target recombination within the host locus.Where constructs are targeted to an endogenous locus, all or some of thetranscriptional and translational regulatory regions can be provided bythe endogenous locus.

Where two or more genes are expressed from separate replicating vectors,it is desirable that each vector has a different means of selection andshould lack homology to the other construct(s) to maintain stableexpression and prevent reassortment of elements among constructs.Judicious choice of regulatory regions, selection means and method ofpropagation of the introduced construct(s) can be experimentallydetermined so that all introduced genes are expressed at the necessarylevels to provide for synthesis of the desired products.

Constructs comprising the gene of interest may be introduced into a hostcell by any standard technique. These techniques include transformation(e.g., lithium acetate transformation [Methods in Enzymology,194:186-187 (1991)]), protoplast fusion, biolistic impact,electroporation, microinjection, or any other method that introduces thegene of interest into the host cell. More specific teachings applicablefor oleaginous yeasts (i.e., Yarrowia lipolytica) include U.S. Pat. Nos.4,880,741 and 5,071,764 and Chen, D. C. et al. (Appl MicrobiolBiotechnol. 48(2):232-235(1997)).

For convenience, a host cell that has been manipulated by any method totake up a DNA sequence (e.g., an expression cassette) will be referredto as “transformed” or “recombinant” herein. The transformed host willhave at least one copy of the expression construct and may have two ormore, depending upon whether the gene is integrated into the genome,amplified or is present on an extrachromosomal element having multiplecopy numbers. The transformed host cell can be identified by selectionfor a marker contained on the introduced construct. Alternatively, aseparate marker construct may be co-transformed with the desiredconstruct, as many transformation techniques introduce many DNAmolecules into host cells. Typically, transformed hosts are selected fortheir ability to grow on selective media. Selective media mayincorporate an antibiotic or lack a factor necessary for growth of theuntransformed host, such as a nutrient or growth factor. An introducedmarker gene may confer antibiotic resistance, or encode an essentialgrowth factor or enzyme, thereby permitting growth on selective mediawhen expressed in the transformed host. Selection of a transformed hostcan also occur when the expressed marker protein can be detected, eitherdirectly or indirectly. The marker protein may be expressed alone or asa fusion to another protein. The marker protein can be detected by: 1.)its enzymatic activity (e.g., β-galactosidase can convert the substrateX-gal [5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside] to a coloredproduct; luciferase can convert luciferin to a light-emitting product);or 2.) its light-producing or modifying characteristics (e.g., the greenfluorescent protein of Aequorea Victoria fluoresces when illuminatedwith blue light). Alternatively, antibodies can be used to detect themarker protein or a molecular tag on, for example, a protein ofinterest. Cells expressing the marker protein or tag can be selected,for example, visually, or by techniques such as FACS or panning usingantibodies. For selection of yeast transformants, any marker thatfunctions in yeast may be used. Desirably, resistance to kanamycin,hygromycin and the amino glycoside G418 are of interest, as well asability to grow on media lacking uracil or leucine.

Following transformation, substrates suitable for the gene products ofthe instant sequences (and optionally other PUFA enzymes that areexpressed within the host cell), may be produced by the host eithernaturally or transgenically, or they may be provided exogenously.

Fermentation Processes for Triacylglycerol Biosynthesis and Accumulation

The transformed microbial host cell is grown under conditions thatoptimize activity of fatty acid biosynthetic genes and acyltransferasegenes. This leads to production of the greatest and the most economicalyield of fatty acids, which can in turn be transferred to TAGs forstorage. In general, media conditions that may be optimized include thetype and amount of carbon source, the type and amount of nitrogensource, the carbon-to-nitrogen ratio, the oxygen level, growthtemperature, pH, length of the biomass production phase, length of theoil accumulation phase and the time of cell harvest. Microorganisms ofinterest, such as oleaginous yeast, are grown in complex media (e.g.,yeast extract-peptone-dextrose broth (YPD)) or a defined minimal mediathat lacks a component necessary for growth and thereby forces selectionof the desired expression cassettes (e.g., Yeast Nitrogen Base (DIFCOLaboratories, Detroit, Mich.)).

Fermentation media in the present invention must contain a suitablecarbon source. Suitable carbon sources may include, but are not limitedto: monosaccharides (e.g., glucose, fructose), disaccharides (e.g.,lactose, sucrose), oligosaccharides, polysaccharides (e.g., starch,cellulose or mixtures thereof), sugar alcohols (e.g., glycerol) ormixtures from renewable feedstocks (e.g., cheese whey permeate,cornsteep liquor, sugar beet molasses, barley malt). Additionally,carbon sources may include alkanes, fatty acids, esters of fatty acids,monoglycerides, diglycerides, triglycerides, phospholipids and variouscommercial sources of fatty acids including vegetable oils (e.g.,soybean oil) and animal fats. Additionally, the carbon substrate mayinclude one-carbon substrates (e.g., carbon dioxide, methanol,formaldehyde, formate, carbon-containing amines) for which metabolicconversion into key biochemical intermediates has been demonstrated.Hence it is contemplated that the source of carbon utilized in thepresent invention may encompass a wide variety of carbon-containingsubstrates and will only be limited by the choice of the host organism.Although all of the above mentioned carbon substrates and mixturesthereof are expected to be suitable in the present invention, preferredcarbon substrates are sugars and/or fatty acids. Most preferred isglucose and/or fatty acids containing between 10-22 carbons.

Nitrogen may be supplied from an inorganic (e.g., (NH₄)₂SO₄) or organicsource (e.g., urea, glutamate). In addition to appropriate carbon andnitrogen sources, the fermentation media must also contain suitableminerals, salts, cofactors, buffers, vitamins and other components knownto those skilled in the art suitable for the growth of the microorganismand promotion of the enzymatic pathways necessary for fatty acidproduction. Particular attention is given to several metal ions (e.g.,Mn⁺², Co⁺², Zn⁺², Mg⁺²) that promote synthesis of lipids and PUFAs(Nakahara, T. et al., Ind. Appl. Single Cell Oils, D. J. Kyle and R.Colin, eds. pp 61-97 (1992)).

Preferred growth media in the present invention are common commerciallyprepared media, such as Yeast Nitrogen Base (DIFCO Laboratories,Detroit, Mich.). Other defined or synthetic growth media may also beused and the appropriate medium for growth of the particularmicroorganism will be known by one skilled in the art of microbiology orfermentation science. A suitable pH range for the fermentation istypically between about pH 4.0 to pH 8.0, wherein pH 5.5 to pH 7.0 ispreferred as the range for the initial growth conditions. Thefermentation may be conducted under aerobic or anaerobic conditions,wherein microaerobic conditions are preferred.

Typically, accumulation of high levels of fatty acids and TAGs inoleaginous yeast cells requires a two-stage process, since the metabolicstate must be “balanced” between growth and synthesis/storage of fats.Thus, most preferably, a two-stage fermentation process is necessary forthe production of oils in oleaginous yeast. In this approach, the firststage of the fermentation is dedicated to the generation andaccumulation of cell mass and is characterized by rapid cell growth andcell division. In the second stage of the fermentation, it is preferableto establish conditions of nitrogen deprivation in the culture topromote high levels of lipid accumulation. The effect of this nitrogendeprivation is to reduce the effective concentration of AMP in thecells, thereby reducing the activity of the NAD-dependent isocitratedehydrogenase of mitochondria. When this occurs, citric acid willaccumulate, thus forming abundant pools of acetyl-CoA in the cytoplasmand priming fatty acid synthesis. Thus, this phase is characterized bythe cessation of cell division followed by the synthesis of fatty acidsand accumulation of TAGs.

Although cells are typically grown at about 30° C., some studies haveshown increased synthesis of unsaturated fatty acids at lowertemperatures (Yongmanitchai and Ward, Appl. Environ. Microbiol.57:419-25 (1991)). Based on process economics, this temperature shiftshould likely occur after the first phase of the two-stage fermentation,when the bulk of the organisms' growth has occurred.

It is contemplated that a variety of fermentation process designs may beapplied, where commercial production of fatty acids and TAGs using theinstant genes is desired. For example, commercial production of TAGscontaining PUFAs from a recombinant microbial host may be produced by abatch, fed-batch or continuous fermentation process.

A batch fermentation process is a closed system wherein the mediacomposition is set at the beginning of the process and not subject tofurther additions beyond those required for maintenance of pH and oxygenlevel during the process. Thus, at the beginning of the culturingprocess the media is inoculated with the desired organism and growth ormetabolic activity is permitted to occur without adding additionalsubstrates (i.e., carbon and nitrogen sources) to the medium. In batchprocesses the metabolite and biomass compositions of the system changeconstantly up to the time the culture is terminated. In a typical batchprocess, cells moderate through a static lag phase to a high-growth logphase and finally to a stationary phase, wherein the growth rate isdiminished or halted. Left untreated, cells in the stationary phase willeventually die. A variation of the standard batch process is thefed-batch process, wherein the substrate is continually added to thefermentor over the course of the fermentation process. A fed-batchprocess is also suitable in the present invention. Fed-batch processesare useful when catabolite repression is apt to inhibit the metabolismof the cells or where it is desirable to have limited amounts ofsubstrate in the media at any one time. Measurement of the substrateconcentration in fed-batch systems is difficult and therefore may beestimated on the basis of the changes of measurable factors such as pH,dissolved oxygen and the partial pressure of waste gases (e.g., CO₂).Batch and fed-batch culturing methods are common and well known in theart and examples may be found in Thomas D. Brock in Biotechnology: ATextbook of Industrial Microbioloqy, 2^(nd) ed., (1989) SinauerAssociates: Sunderland, Mass.; or Deshpande, Mukund V., Appl. Biochem.Biotechnol., 36:227 (1992), herein incorporated by reference.

Commercial production of fatty acids using the instant genes may also beaccomplished by a continuous fermentation process wherein a definedmedia is continuously added to a bioreactor while an equal amount ofculture volume is removed simultaneously for product recovery.Continuous cultures generally maintain the cells in the log phase ofgrowth at a constant cell density. Continuous or semi-continuous culturemethods permit the modulation of one factor or any number of factorsthat affect cell growth or end product concentration. For example, oneapproach may limit the carbon source and allow all other parameters tomoderate metabolism. In other systems, a number of factors affectinggrowth may be altered continuously while the cell concentration,measured by media turbidity, is kept constant. Continuous systems striveto maintain steady state growth and thus the cell growth rate must bebalanced against cell loss due to media being drawn off the culture.Methods of modulating nutrients and growth factors for continuousculture processes, as well as techniques for maximizing the rate ofproduct formation, are well known in the art of industrial microbiologyand a variety of methods are detailed by Brock, supra.

Purification of Fatty Acids

Fatty acids, including PUFAs, may be found in the host microorganism asfree fatty acids or in esterified forms such as acylglycerols,phospholipids, sulfolipids or glycolipids, and may be extracted from thehost cell through a variety of means well-known in the art. One reviewof extraction techniques, quality analysis and acceptability standardsfor yeast lipids is that of Z. Jacobs (Critical Reviews in Biotechnology12(5/6):463-491 (1992)). A brief review of downstream processing is alsoavailable by A. Singh and O. Ward (Adv. Appl. Microbiol. 45:271-312(1997)).

In general, means for the purification of fatty acids, including PUFAs,may include extraction with organic solvents, sonication, supercriticalfluid extraction (e.g., using carbon dioxide), saponification andphysical means such as presses, or combinations thereof. Of particularinterest is extraction with methanol and chloroform in the presence ofwater (E. G. Bligh & W. J. Dyer, Can. J. Biochem. Physiol. 37:911-917(1959)). Where desirable, the aqueous layer can be acidified toprotonate negatively-charged moieties and thereby increase partitioningof desired products into the organic layer. After extraction, theorganic solvents can be removed by evaporation under a stream ofnitrogen. When isolated in conjugated forms, the products may beenzymatically or chemically cleaved to release the free fatty acid or aless complex conjugate of interest, and can then be subject to furthermanipulations to produce a desired end product. Desirably, conjugatedforms of fatty acids are cleaved with potassium hydroxide.

If further purification is necessary, standard methods can be employed.Such methods may include extraction, treatment with urea, fractionalcrystallization, HPLC, fractional distillation, silica gelchromatography, high-speed centrifugation or distillation, orcombinations of these techniques. Protection of reactive groups, such asthe acid or alkenyl groups, may be done at any step through knowntechniques (e.g., alkylation, iodination). Methods used includemethylation of the fatty acids to produce methyl esters. Similarly,protecting groups may be removed at any step. Desirably, purification offractions containing GLA, STA, ARA, DHA and EPA may be accomplished bytreatment with urea and/or fractional distillation.

DESCRIPTION OF PREFERRED EMBODIMENTS

The ultimate goal of the work described herein is the development of anoleaginous yeast that accumulates TAGs enriched in ω-3 and/or ω-6 PUFAs.Toward this end, acyltransferases must be identified that functionefficiently in oleaginous yeasts, to enable synthesis and highaccumulation of preferred TAGs in these hosts. Specifically,modification of the expression levels of these acyltransferases willenable increased transfer of fatty acids (and particularly, PUFAs) toTAGs. Thus, identification of efficient acyltransferases is necessaryfor the manipulation of the amount of ω-3/ω-6 PUFAs incorporated intothe TAG fraction produced in host cells.

In the present invention, Applicants have isolated and cloned genes fromYarrowia lipolytica that encode PDAT and DGAT2. Confirmation of thesegenes' activity was provided based upon lower oil content (total fattyacids as a % of dry cell weight) in Yarrowia strains wherein disruptionof the native PDAT, DGAT2, or PDAT and DGAT2 had occurred by targetedgene replacement through homologous recombination (Example 5).Additionally, over-expression of the PDAT of the invention in aPDAT/DGAT2 knockout strain of Saccharomyces cerevisiae lead to increasedoil content (total fatty acids as a % of dry cell weight).

The Applicants conclude that these acyltransferase genes encoding PDATand DGAT2 are useful for expression in various microbial hosts, andparticularly for over-expression in oleaginous yeasts (e.g., the nativehost Yarrowia lipolytica). Additional benefits may result, sinceexpression of the acyltransferases can also be put under the control ofstrong constitutive or regulated promoters that do not have theregulatory constraints of the native gene.

EXAMPLES

The present invention is further defined in the following Examples. Itshould be understood that these Examples, while indicating preferredembodiments of the invention, are given by way of illustration only.From the above discussion and these Examples, one skilled in the art canascertain the essential characteristics of this invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usages andconditions.

General Methods

Standard recombinant DNA and molecular cloning techniques used in theExamples are well known in the art and are described by: 1.) Sambrook,J., Fritsch, E. F. and Maniatis, T. Molecular Cloning: A LaboratoryManual; Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y. (1989)(Maniatis); 2.) T. J. Silhavy, M. L. Bennan, and L. W. Enquist,Experiments with Gene Fusions; Cold Spring Harbor Laboratory: ColdSpring Harbor, N.Y. (1984); and 3.) Ausubel, F. M. et al., CurrentProtocols in Molecular Biology, published by Greene Publishing Assoc.and Wiley-Interscience (1987).

Materials and methods suitable for the maintenance and growth ofmicrobial cultures are well known in the art. Techniques suitable foruse in the following Examples may be found as set out in Manual ofMethods for General Bacteriology (Phillipp Gerhardt, R. G. E. Murray,Ralph N. Costilow, Eugene W. Nester, Willis A. Wood, Noel R. Krieg andG. Briggs Phillips, Eds), American Society for Microbiology: Washington,D.C. (1994)); or by Thomas D. Brock in Biotechnoloqy: A Textbook ofIndustrial Microbioloqy, 2^(nd) ed., Sinauer Associates: Sunderland,Mass. (1989). All reagents, restriction enzymes and materials used forthe growth and maintenance of microbial cells were obtained from AldrichChemicals (Milwaukee, Wis.), DIFCO Laboratories (Detroit, Mich.),GIBCO/BRL (Gaithersburg, Md.) or Sigma Chemical Company (St. Louis,Mo.), unless otherwise specified.

E. coli TOP10 cells and E. coli Electromax DH10B cells were obtainedfrom Invitrogen (Carlsbad, Calif.). Max Efficiency competent cells of E.coli DH5α were obtained from GIBCO/BRL (Gaithersburg, Md.). E. coli(XL1-Blue) competent cells were purchased from the Stratagene Company(San Diego, Calif.). E. coli strains were typically grown at 37° C. onLuria Bertani (LB) plates. General molecular cloning was performedaccording to standard methods (Sambrook et al., supra). Oligonucleotideswere synthesized by Sigma-Genosys (Spring, Tex.). PCR products werecloned into Promega's pGEM-T-easy vector (Madison, Wis.).

DNA sequence was generated on an ABI Automatic sequencer using dyeterminator technology (U.S. Pat. No. 5,366,860; EP 272,007) using acombination of vector and insert-specific primers. Sequence editing wasperformed in Sequencher (Gene Codes Corporation, Ann Arbor, Mich.). Allsequences represent coverage at least two times in both directions.Comparisons of genetic sequences were accomplished using DNASTARsoftware (DNASTAR, Inc., (Madison, Wis.).

The meaning of abbreviations is as follows: “sec” means second(s), “min”means minute(s), “h” means hour(s), “d” means day(s), “μL” meansmicroliter(s), “mL” means milliliter(s), “L” means liter(s), “μM” meansmicromolar, “mM” means millimolar, “M” means molar, “mmol” meansmillimole(s), “μmole” mean micromole(s), “g” means gram(s), “μg” meansmicrogram(s), “ng” means nanogram(s), “U” means unit(s), “bp” means basepair(s) and “kB” means kilobase(s).

Cultivation of Yarrowia lipolytica

Yarrowia lipolytica strains ATCC #76982 and ATCC #90812 were purchasedfrom the American Type Culture Collection (Rockville, Md.). Y.lipolytica strains were usually grown at 28° C. on YPD agar (1% yeastextract, 2% bactopeptone, 2% glucose, 2% agar). For selection oftransformants, minimal medium (0.17% yeast nitrogen base (DIFCOLaboratories, Detroit, Mich.) without ammonium sulfate or amino acids,2% glucose, 0.1% proline, pH 6.1) was used. Supplements of adenine,leucine, lysine and/or uracil were added as appropriate to a finalconcentration of 0.01%.

Fatty Acid Analysis of Yarrowia lipolytica

For fatty acid analysis, cells were collected by centrifugation andlipids were extracted as described in Bligh, E. G. & Dyer, W. J. (Can.J. Biochem. Physiol. 37:911-917 (1959)). Fatty acid methyl esters wereprepared by transesterification of the lipid extract with sodiummethoxide (Roughan, G., and Nishida I. Arch Biochem Biophys. 276(1):3846(1990)) and subsequently analyzed with a Hewlett-Packard 6890 GC fittedwith a 30-m×0.25 mm (i.d.) HP-INNOWAX (Hewlett-Packard) column. The oventemperature was from 170° C. (25 min hold) to 185° C. at 3.5° C./min.

For direct base transesterification, Yarrowia culture (3 mL) washarvested, washed once in distilled water and dried under vacuum in aSpeed-Vac for 5-10 min. Sodium methoxide (100 μl of 1%) was added to thesample, and then the sample was vortexed and rocked for 20 min. Afteradding 3 drops of 1 M NaCl and 400 μl hexane, the sample was vortexedand spun. The upper layer was removed and analyzed by GC as describedabove.

EXAMPLE 1 Construction of Plasmids Suitable for Gene Expression inYarrowia lipolytica

The present Example describes the construction of plasmids pY5, pY5-13,pY5-20 and pLV5.

Construction of Plasmid pY5

The plasmid pY5, a derivative of pINA532 (a gift from Dr. ClaudeGaillardin, Insitut National Agronomics, Centre de biotechnologieAgro-Industrielle, laboratoire de Genetique Moleculaire et CellularieINRA-CNRS, F-78850 Thiverval-Grignon, France), was constructed forexpression of heterologous genes in Yarrowia lipolytica, as diagrammedin FIG. 3. First, the partially-digested 3598 bp EcoRi fragmentcontaining the ARS18 sequence and LEU2 gene of pINA532 was subclonedinto the EcoRi site of pBluescript (Strategene, San Diego, Calif.) togenerate pY2. The TEF promoter (Muller S., et al., Yeast, 14:1267-1283(1998)) was amplified from Y. lipolytica genomic DNA by PCR using TEF5′(SEQ ID NO:1) and TEF3′ (SEQ ID NO:2) as primers. PCR amplification wascarried out in a 50 μl total volume containing: 100 ng Yarrowia genomicDNA, PCR buffer containing 10 mM KCl, 10 mM (NH₄)₂SO₄, 20 mM Tris-HCl(pH 8.75), 2 mM MgSO₄, 0.1% Triton X-100, 100 μg/mL BSA (finalconcentration), 200 μM each deoxyribonucleotide triphosphate, 10 pmoleof each primer and 1 μl of Pfu Turbo DNA polymerase (Stratagene).Amplification was carried out as follows: initial denaturation at 95° C.for 3 min, followed by 35 cycles of the following: 95° C. for 1 min, 56°C. for 30 sec, 72° C. for 1 min. A final extension cycle of 72° C. for10 min was carried out, followed by reaction termination at 4° C. The418 bp PCR product was ligated into pCR-Blunt to generate pIP-tef. TheBamHI/EcoRV fragment of pIP-tef was subcloned into the BamHI/Smal sitesof pY2 to generate pY4.

The XPR2 transcriptional terminator was amplified by PCR using pINA532as template and XPR5′ (SEQ ID NO:3) and XPR3′ (SEQ ID NO:4) as primers.The PCR amplification was carried out in a 50 μl total volume, using thecomponents and conditions described above. The 179 bp PCR product wasdigested with SacII and then ligated into the SacII site of pY4 togenerate pY5. Thus, pY5 (shown in FIG. 3) is useful as a Yarrowia-E.coli shuttle plasmid containing:

-   -   1.) a Yarrowia autonomous replication sequence (ARS18);    -   2.) a ColE1 plasmid origin of replication;    -   3.) an ampicillin-resistance gene (AmPR), for selection in E.        coli;    -   4.) a Yarrowia LEU2 gene (E.C. 1.1.1.85, encoding        isopropylmalate isomerase), for selection in Yarrowia;    -   5.) the translation elongation promoter (TEF), for expression of        heterologous genes in Yarrowia; and    -   6.) the extracellular protease gene terminator (XPR2) for        transcriptional termination of heterologous gene expression in        Yarrowia.        Construction of Plasmid pY5-13

pY5-13 (FIG. 3) was constructed as a derivative of pY5 to faciliatesubcloning and heterologous gene expression in Yarrowia lipolytica.Specifically, pY5-13 was constructed by 6 rounds of site-directedmutagenesis using pY5 as template. Both SaII and ClaI sites wereeliminated from pY5 by site-directed mutagenesis using oligonucleotidesYL5 and YL6 (SEQ ID NOs:5 and 6) to generate pY5-5. A SaII site wasintroduced into pY5-5 between the LEU2 gene and the TEF promoter bysite-directed mutagenesis using oligonucleotides YL9 and YL10 (SEQ IDNOs:7 and 8) to generate pY5-6. A PacI site was introduced into pY5-6between the LEU2 gene and ARS18 using oligonucleotides YL7 and YL8 (SEQID NOs:9 and 10) to generate pY5-8. A NcoI site was introduced intopY5-8 around the translation start codon of the TEF promoter usingoligonucleotides YL3 and YL4 (SEQ ID NOs:11 and 12) to generate pY5-9.The Ncol site inside the LEU2 gene of pY5-9 was eliminated using YL1 andYL2 oligonucleotides (SEQ ID NOs:13 and 14) to generate pY5-12. Finally,a BsiWI site was introduced into pY5-12 between the ColEI and XPR2region using oligonucleotides YL61 and YL62 (SEQ ID NOs:15 and 16) togenerate pY5-13.

Construction of Plasmids pY5-20 and pLV5

Plasmid pY5-20 is a derivative of pY5. It was constructed by inserting aNot I fragment containing a chimeric hygromycin resistance gene into theNot I site of pY5. Specifically, the E. coli hygromycin resistance gene(SEQ ID NO:17; “HPT”; Kaster, K. R., et al., Nucleic Acids Res.11:6895-6911 (1983)) was PCR amplified for expression. The chimeric genehad the hygromycin resistance ORF under the control of the Y. lipolyticaTEF promoter.

Plasmid pLV5 is a derivative of pY5-20. It was constructed by replacingthe hygromycin resistant gene with the Yarrowia Ura3 gene. A 1.7 kB DNAfragment (SEQ ID NO:19) containing the Yarrowia Ura3 gene was PCRamplified using oligonucleotides KU5 and KU3 (SEQ ID NOs:21 and 22) asprimers and Yarrowia genomic DNA as template.

EXAMPLE 2 Cloning of a Partial Yarrowia lipolyticaAcyl-CoA:Diacylglycerol Acyltransferase (DGAT2) Gene and Disruption ofThe Endogenous DGAT2 Gene

The present Example describes the use of degenerate PCR primers toisolate a partial coding sequence of the Yarrowia lipolytica DGAT2 andthe use of the partial sequence to disrupt the native gene in Y.lipolytica.

Cloning of a Partial Putative DGAT2 Sequence from Yarrowia lipolytica byPCR Using Degenerate PCR Primers and Chromosome Walking

Genomic DNA was isolated from Y. lipolytica (ATCC #76982) using a DNeasyTissue Kit (Qiagen, Catalog #69504) and resuspended in kit buffer AE ata DNA concentration of 0.5 μg/μl. PCR amplifications were performedusing the genomic DNA as template and several sets of degenerate primersdesigned to encode conserved amino acid sequences among different knownDGAT2s (i.e., GenBank Accession Nos. NC_(—)001147 [Saccharomycescerevisiae] and AF391089 and AF391090 [Mortierella ramanniana]). Thebest results were obtained with degenerate primers P7 and P8, as shownin the Table below. TABLE 3 Degenerate Primers Used For Amplification OfA Partial Putative DGAT2 Corresponding Primer Amino Acid Set DescriptionDegenerate Nucleotide Sequence Sequence P7 (32) 29-mers 5′- NYIFGYHPHGAACTACATCTTCGGCTAY (SEQ ID NO:24) CAYCCNCAYGG-3′ (SEQ ID NO:23) P8 (48)29-mers 5′- complementary to AGGGACTCGGAGGCGC IVVGGASESLCGCCNCANACDAT-3′ (SEQ ID NO:26) (SEQ ID NO:25)[Note:Abbreviations are standard for nucleotides and proteins. The nucleicacid degeneracy code used is as follows: Y = C/T; D = A/G/T; and N= A/C/G/T.]

The PCR was carried out in a RoboCycler Gradient 40 PCR machine(Stratagene) using the manufacturer's recommendations and Accuprime Taqpolymerase (Invitrogen). Amplification was carried out as follows:initial denaturation at 95° C. for 1 min, followed by 30 cycles ofdenaturation at 95° C. for 30 sec, annealing at 55° C. for 1 min, andelongation at 72° C. for 1 min. A final elongation cycle at 72° C. for10 min was carried out, followed by reaction termination at 4° C.

The expected PCR product (ca. 264 bp) was detected by 4% NuSieve (FMC)agarose gel electrophoresis, isolated, purified, cloned into the TOPO®cloning vector (Invitrogen), and sequenced. The resultant sequence(contained within SEQ ID NO:30) had homology to known DGAT2s, based onBLAST program analysis (Basic Local Alignment Search Tool; Altschul, S.F., et al., J. Mol. Biol. 215:403-410 (1993).

Using the 264 bp fragment as an initiation point, a 673 bp fragment wasobtained by chromosome walking using the TOPO® Walker Kit (Invitrogen,Catalog #K8000-01). The chromosome walking was carried out in 6 steps,as described briefly below:

-   -   1.) Genomic DNA (5 μg) was digested with restriction enzymes Pst        I or Sac I, leaving a 3′ overhang;    -   2.) Digested DNA was treated with 0.1 U calf intestinal alkaline        phosphatase to dephosphorylate DNA;    -   3.) Primer extension was performed, using the DGAT2 specific        primer P80 (SEQ ID NO:27) and Taq polymerase;    -   4.) TOPO® Linker (1 μl) was added and the reaction was incubated        at 37° C. for 5 min to ligate TOPO® Linker to the DNA;    -   5.) PCR was performed using the DGAT2 gene specific primer, P81        (SEQ ID NO:28) and LinkAmp primer 1 (SEQ ID NO:29); and    -   6.) The newly amplified fragment was sequenced with primer P81        and LinkAmp primer 1.        The sequence of the 673 bp fragment obtained by chromosome        walking also showed homology to known DGAT2 sequences.        Targeted Disruption of the Yarrowia lipolytica DGAT2 Gene

Targeted disruption of the DGAT2 gene in Y. lipolytica ATCC #90812 andATCC #76982 was carried out by homologous recombination-mediatedreplacement of the endogenous DGAT2 gene with a targeting cassettedesignated as plasmid pY21 DGAT2. pY21 DGAT2 was derived from plasmidpY20 (Example 1). Specifically, pY21 DGAT2 was created by inserting a570 bp Hind III/Eco RI fragment into similarly linearized pY20. The 570bp DNA fragment contained (in 5′ to 3′ orientation): 3′ homologoussequence from position +1090 to +1464 (of the coding sequence (ORF) inSEQ ID NO:30), a Bgl II restriction site and 5′ homologous sequence fromposition +906 to +1089 (of the coding sequence (ORF) shown in SEQ IDNO:30). The fragment was prepared by PCR amplification of 3′ and 5′sequences from the 673 bp DGAT2 PCR product obtained by chromosomewalking using two pairs of PCR primers, P95 and P96 (SEQ ID NOs:32 and33), and P97 and P98 (SEQ ID NOs:34 and 35), respectively.

pY21 DGAT2 was linearized by Bgl II restriction digestion andtransformed into mid-log phase Y. lipolytica ATCC #90812 and ATCC #76982cells by the lithium acetate method according to the method of Chen, D.C. et al. (Appl Microbiol Biotechnol. 48(2):232-235-(1997)). Briefly, Y.lipolytica ATCC #90821 and Y. lipolytica ATCC #76982 were streaked ontoYPD plates and grown at 30° C. for approximately 18 hr. Several largeloopfuls of cells were scraped from the plates and resuspended in 1 mLof transformation buffer containing:

-   -   2.25 mL of 50% PEG, average MW 3350;    -   0.125 mL of 2 M Li acetate, pH 6.0;    -   0.125 mL of 2 M DTT; and    -   50 μg sheared salmon sperm DNA.        About 500 ng of plasmid DNA were incubated in 100 μl of        resuspended cells and maintained at 39° C. for 1 hr with vortex        mixing at 15 min intervals. The cells were plated onto YPD        hygromycin selection plates and maintained at 30° C. for 2 to 3        days.

Four Y. lipolytica ATCC #76982 hygromycin-resistant colonies andfourteen Y. lipolytica ATCC #90812 hygromycin-resistant colonies wereisolated and screened for targeted disruption by PCR. One set of PCRprimers (P115 [SEQ ID NO:36] and P116 [SEQ ID NO:37]) was designed toamplify a specific junction fragment following homologous recombination.Another pair of PCR primers (P115 and P112 [SEQ ID NO:38]) was designedto detect the native gene. All (4 of 4) of the hygromycin-resistantcolonies of ATCC #76982 strains were positive for the junction fragmentand negative for the native fragment; and, 2 of the 14hygromycin-resistant colonies of ATCC #90812 strains were positive forthe junction fragment and negative for the native fragment. Thus,targeted integration was confirmed in these 6 strains. Disruption of thegene was further confirmed by GC analysis of total lipids of one of thedisrupted strains, designated as “S-D” (see Example 5).

EXAMPLE 3 Cloning of a Partial Yarrowia lipolyticaPhospholipid:Diacylglycerol Acyltransferase (PDAT) Gene and Disruptionof the Endogenous PDAT Gene

The present Example describes the use of degenerate PCR primers toisolate a partial coding sequence of Y. lipolytica PDAT and the use ofthe partial sequence to disrupt the native gene in Y. lipolytica.

Cloning of a Partial Putative PDAT Sequence from Yarrowia lipolytica byPCR Using Degenerate PCR Primers and Chromosome Walking

Genomic DNA was isolated from Y. lipolytica (ATCC #76982) using a DNeasyTissue Kit (Qiagen, Catalog # 69504) and resuspended in kit buffer AE ata DNA concentration of 0.5 μg/μl. PCR amplifications were performedusing genomic DNA as the template and several pairs of degenerateprimers encoding conserved amino acid sequences in different known PDATs(GenBank Accession Nos. NP 190069 and AB006704 [(gi:2351069 Arabidopsisthaliana], and NP_(—)596330 [Schizosaccharomyces pombe]; and theSaccharomyces cerevisiae Lro 1 gene [Dahlqvist et al., Proc. Natl. Acad.Sci. USA 97:6487 (2000)]). The best results were obtained withdegenerate primers P26 and P27, as shown in the Table below. TABLE 4Degenerate Primers Used For Amplification Of A Partial Putative PDATCorresponding Primer Amino Acid Set Description Degenerate NucleotideSequence Sequence P26 (32) 29-mers 5′- MLDKETGLDP ATGCTGGACAAGGAGACCGGNC(SEQ ID NO:40) TNGAYCC-3′ (SEQ ID NO:39) P27 (16) 33-mers 5′-complementary to CCAGATGACGTCGCCGCCCTTG SMLPKGGEVIW GGNARCATNGA-3′ (SEQID NO:42) (SEQ ID NO:41)[Note:Abbreviations are standard for nucleotides and proteins. The nucleicacid degeneracy code used is as follows: R = A/G; Y = C/T; and N= A/C/G/T.]

The PCR was carried out in a RoboCycler Gradient 40 PCR machine(Stratagene), using the amplification conditions described in Example 2.The expected PCR product (ca. 600 bp) was detected by 4% NuSieve (FMC)agarose gel electrophoresis, isolated, purified, cloned into the TOPO®cloning vector (Invitrogen) and sequenced. The resultant sequence(contained within SEQ ID NO:45) had homology to known PDATs, based onBLAST program analysis (Basic Local Alignment Search Tool; Altschul, S.F., et al., J. Mol. Biol. 215:403-410 (1993).

Targeted Disruption of Yarrowia lipolytica PDAT Gene

Following the sequencing of this ca. 600 bp partial coding region forPDAT, a larger DNA fragment encoding this sequence was discovered in thepublic Y. lipolytica database of the “Yeast project Genolevures” (Centerfor Bioinformatics, LaBRI, Talence Cedex, France. This allowed isolationof a 1008 bp genomic DNA fragment comprising a portion of the PDAT genefrom Y. lipolytica ATCC #90812 using PCR primers P39 and P42 (SEQ IDNOs:43 and 44).

Targeted disruption of the PDAT gene in Y. lipolytica ATCC #90812 wascarried out by homologous recombination-mediated replacement of theendogenous PDAT gene with a targeting cassette designated as pLV13.pLV13 was derived from plasmid pLV5 (Example 1). Specifically, pLV13 wascreated by inserting a 992 bp Bam HI/Eco RI fragment into similarlylinearized pLV5. The 992 bp DNA fragment contained (in 5′ to 3′orientation): 3′ homologous sequence from position +877 to +1371 (of thecoding sequence (ORF) in SEQ ID NO:45), a Bgl II restriction site and 5′homologous sequence from position +390 to +876 (of the coding sequence(ORF) in SEQ ID NO:45). The fragment was prepared by PCR amplificationof 3′ and 5′ sequences from the 1008 bp PCR product described above,using PCR primers P39 and P41 (SEQ ID NOs:43 and 47) and P40 and P42(SEQ ID NOs:48 and 44), respectively.

pLV13 was linearized by Bgl II restriction digestion and was transformedinto mid-log phase Y. lipolytica ATCC #90812 cells by the lithiumacetate method (Example 2). The cells were plated onto Bio101DOB/CSM-Ura selection plates and maintained at 30° C. for 2 to 3 days.

Ten Y. lipolytica ATCC #90812 colonies were isolated and screened fortargeted disruption by PCR. One set of PCR primers (P51 [SEQ ID NO:49]and P52 [SEQ ID NO:50]) was designed to amplify a targeting cassette.Another set of PCR primers (P37 [SEQ ID NO:51] and P38 [SEQ ID NO:52])was designed to detect the native gene. Ten of the ten strains werepositive for the junction fragment and 3 of the 10 strains were negativefor native fragment, thus confirming successful targeted integration inthese 3 strains. Disruption of the gene was further confirmed by GCanalysis of total lipids in one of the disrupted strains, designated as“S-P” (see Example 5).

EXAMPLE 4 Construction of a Yarrowia lipolytica Double Knockout StrainContaining Disruptions in Both PDAT and DGAT2 Genes

The present Example describes the creation of a double knockout strainthat was disrupted in both PDAT and DGAT2 genes.

Specifically, the Y. lipolytica ATCC#90812 hygromycin-resistant “S-D”mutant (containing the DGAT2 disruption from Example 2) was transformedwith plasmid pLV13 (from Example 3) and transformants were screened byPCR; as described in Example 3. Two of twelve transformants wereconfirmed to be disrupted in both the DGAT2 and PDAT genes. Disruptionof the gene was further confirmed by GC analysis of total lipids in oneof the disrupted strains, designated as “S-D-P” (see Example 5).

EXAMPLE 5 Determination of TAG Content in Mutant and Wildtype Yarrowialipolytica Strains (ATCC #90812)

Single colonies of wildtype and mutant Y. lipolytica (ATCC #90812)containing disruptions in either the PDAT (from Example 3), DGAT2 (fromExample 2) or PDAT and DGAT2 (from Example 4) genes were separatelygrown according to two different culture conditions, as described below:

-   -   Growth Condition 1: Cells were grown in 3 mL minimal media        (formulation/L: 20 g glucose, 1.7 g yeast nitrogen base, 1 g        L-proline, 0.1 g L-adenine, 0.1 g L-lysine, pH 6.1) at 30° C. to        an OD₆₀₀ ˜1.0. The cells were harvested, washed in distilled        water, speed vacuum dried and subjected to GC analysis of the        lipids following thin layer chromatography (TLC) (infra).    -   Growth Condition 2: Cells were grown in a 50 mL culture using        conditions that induce oleaginy. Specifically, one loopful of        cells from plates were inoculated into 3 mL YPD medium and grown        overnight on a shaker (300 rpm) at 30° C. The cells were        harvested and washed once in 0.9% NaCl and resuspended in 50 mL        of high glucose medium [formulation/L: 7 g KH₂PO₄, 2 g K₂HPO₄, 2        g MgSO₄.7H₂O, 80 g glucose, 0.1 g leucine, 0.1 g Uracil, and 0.1        g L-lysine, pH 5.0]. Cells were then grown on a shaker as above        for 48 hrs. Cells were washed in water and the cell pellet was        lophilized. Twenty (20) mg of dry cell weight was used for total        fafty acid by GC analysis and the oil fraction following TLC        (infra) and GC analysis.        Thin Layer Chromatography

The methodology used for TLC is described below in the following fivesteps:

-   -   1 ) The internal standard of 15:0 fafty acid (10 μl of 10 mg/mL)        was added to 2 to 3 mg dry cell mass, followed by extraction of        the total lipid using a methanol/chloroform method.    -   2) Extracted lipid (50 μl) was blotted across a light pencil        line drawn approximately 1 inch from the bottom of a 5×20 cm        silica gel 60 plate, using 25-50 μl micropipettes.    -   3) The TLC plate was then dried under N₂ and was inserted into a        tank containing about ˜100 mL 80:20:1 hexane:ethyl ether:acetic        acid solvent.    -   4) After separation of bands, a vapor of iodine was blown over        one side of the plate to identify the bands. This permitted        samples on the other side of the plate to be scraped using a        razor blade for further analysis.    -   5) Basic transesterification of the scraped samples and GC        analysis was performed, as described in the General Methods.        Results from GC Analysis

GC results are shown below in Tables 5 and 6. Cultures are described asthe “S” strain (wildtype), “S-P” (PDAT knockout), “S-D” (DGAT2knockout), and “S-P-D” (PDAT and DGAT2 knockout). Abbreviations utilizedare: WT=wildtype; TFAs=total fafty acids; dcw=dry cell weight; and, %WT=% relative to the wild type (“S” strain). TABLE 5 Lipid Content InYarrowia ATCC #90812 Strains Disrupted In PDAT, DGAT2 Or Both, Grown InMinimal Media TFAs Culture Fraction % dcw % WT S strain (WT) total 12100 TAG 15 100 phospholipid 5 S-P total 11 89 TAG 14 98 phospholipid 5S-D total 10 81 TAG 10 66 phospholipid 4 S-P-D total 8 64 TAG 7 50phospholipid 3

TABLE 6 Lipid Content In Yarrowia ATCC #90812 Strains Disrupted In PDAT,DGAT2 Or Both, Grown Under Oleaginous Conditions TFAs dcw, Lipid % %Culture mg fraction μg dcw WT S strain (WT) 32.0 Total 797 15.9 100 S-D37.5 Total 329 6.4 40 S-P 28.8 Total 318 6.0 38 S-P-D 31.2 Total 228 4.327 S strain (WT) 32.0 TAG 697 13.9 100 S-D 37.5 TAG 227 4.4 32 S-P 28.8TAG 212 4.0 29 S-P-D 31.2 TAG 122 2.3 17The results shown above indicated that the disrupted strains showedlower oil content (TFAs % dcw) as compared to the wild type strain. And,the results shown in Tables 5 and 6 confirmed that the Y. lipolyticagenes encoding both DGAT2 and PDAT contribute to oil biosynthesis in thenative organism, with DGAT2 acting as the major contributor to oilbiosynthesis during oleaginy. Surprisingly, however, the results alsosuggest the existence of additional Yarrowia gene(s) involved in oilbiosynthesis.

EXAMPLE 6 Cloning of Full-Length Yarrowia lipolytica DGAT2 and PDATGenes

The present Example describes the recovery of the genomic sequencesflanking the disrupted DGAT2 and PDAT genes by plasmid rescue, using thesequence in the rescued plasmid to PCR the intact ORF of the nativegene. The full-length genes and their deduced amino acid sequences arecompared to other fungal DGAT2 and PDAT sequences, respectively.

Plasmid Rescue of Yarrowia lipolytica DGAT2 and PDAT Genes

Since the acyltransferase genes were disrupted by the insertion of theentire pY21 DGAT2 and pLV13 vectors that each contained an E. coliampicillin-resistant gene and E. coli ori, it was possible to rescue theflanking PDAT and DGAT2 sequences in E. coli. For this, genomic DNA ofY. lipolytica strain “S-D” (carrying the disrupted DGAT2 gene; Example2) and Y. lipolytica strain “S-P” (carrying the disrupted PDAT gene;Example 3) was isolated using the DNeasy Tissue Kit. Specifically, 10 μgof the genomic DNA was digested with 50 U of the following restrictionenzymes in a reaction volume of 200 μl: for DGAT2—Age I and Nhe I; forPDAT—Kpn I, Pac I and Sac I. Digested DNA was extracted withphenol:chloroform and resuspended in 40 μl deionized water. The digestedDNA (10 μl) was self-ligated in a 200 μl ligation mixture containing 3 UT4 DNA ligase. Each ligation reaction was carried out at 16° C. for 12hrs. The ligated DNA was extracted with phenol:chloroform andresuspended in 40 μl deionized water. Finally, 1 μl of the resuspendedligated DNA was used to transform E. coli by electroporation and platedon LB containing ampicillin (Ap). Ap-resistant transformants wereisolated and analyzed for the presence of plasmids. The following insertsizes were found in the recovered or rescued plasmids (Tables 7 and 8):TABLE 7 Insert Sizes Of Recovered DGAT2 Plasmids, According ToRestriction Enzyme Enzyme plasmid insert size (kB) AgeI 2.3 NheI 9.5

TABLE 8 Insert Sizes Of Recovered PDAT Plasmids, According ToRestriction Enzyme Enzyme plasmid insert size (kB) Kpn I 6.9 Sac I 5.4Sph I 7.0

Sequencing of the DGAT2 rescued plasmids was initiated with sequencingprimers P79 (SEQ ID NO:53) and P95 (SEQ ID NO:32). In contrast,sequencing of the PDAT plasmids was initiated with sequencing primersP84 (SEQ ID NO:54) and P85 (SEQ ID NO:55).

Based on the sequencing results, a full-length gene encoding the Y.lipolytica DGAT2 gene was assembled (2119 bp; SEQ ID NO:30).Specifically, the sequence encoded an open reading frame (ORF) of 1545bases (nucleotides +291 to +1835 of SEQ ID NO:30), while the deducedamino acid sequence was 514 residues in length (SEQ ID NO:31). Sincethis ORF has an initiation codon (‘ATG’) at position 1, as well as atpositions 56 and 160, it contains at least two additional nested(smaller) ORFs. Specifically, one ORF is 1380 bases long (nucleotides+456 to +1835 of SEQ ID NO:30), with a deduced amino acid sequence of459 residues (SEQ ID NO:78); another ORF is 1068 bases long (nucleotides+768 to +1835 of SEQ ID NO:30) with a deduced amino acid sequence of 355residues (SEQ ID NO:79), encoded by SEQ ID NO:86.

The ORF encoded by SEQ ID NO:86 has a high degree of similarity to otherknown DGAT enzymes and because disruption in SEQ ID NO;86 eliminatedDGAT function of the native gene, the polypeptide of SEQ ID NO:79 hasbeen identified as clearly having DGAT functionality. For example, theYarrowia lipolytica DGAT2 that is 355 residues in length (i.e., SEQ IDNO:79) is only 16 amino acids shorter than the Saccharomyces cerevisiaeprotein, 7 amino acids shorter than the Mortierella ramanniana type 2Aprotein and 2 amino acids shorter than the M. ramanniana type 2B protein(infra). Despite this hypothesis, however, it may be useful to test thecontribution of all of the three ORFs encoded by SEQ ID NOs:31, 78 and79 for expression of the Yarrowia DGAT2 protein.

A comparison of SEQ ID NO:79 (i.e., the deduced amino acid sequence ofthe 355 residues; Y. lipolytica DGAT2 (“Yl”)) was made with the knownfungal DGAT2s shown in Table 9 below, using the ClustalW (Slow/Accurate,Gonnet) program of the DNASTAR software package (Madison, Wis.). TABLE 9Description of Known Fungal DGAT2s Organism Abbreviation ReferenceSaccharomyces cerevisiae Sc GenBank Accession No. DGA1 gene NC_001147[Locus NP_014888] Mortierella ramanniana MrA GenBank Accession No. DGAT2type 2A AF391089 Mortierella ramanniana MrB GenBank Accession No. DGAT2type 2B AF391090

This comparison revealed the Pair Distances shown as percent similarityin FIG. 4A. Thus, comparison of the deduced amino acid sequences ofother fungal homologs to the Y. lipolytica DGAT2 described herein as SEQID NO:79 revealed less than 38.4% amino acid identity.

Following sequencing and analysis of the DGAT2 proteins described above,a Yarrowia lipolytica DGAT2 protein sequence was published as part ofthe Genolevures project (sponsored by the Center for Bioinformatics,LaBRI, bâtiment A30, Université Bordeaux 1, 351, cours de la Libération,33405 Talence Cedex, France. Specifically, the sequence disclosedtherein was identified as ORF YALI-CDS2240.1, encoding 514 amino acids,and the protein was reported to share some similarities with tr|Q08650Saccharomyces cerevisiae YOR245C DGA1 acyl-CoA:diacylglycerolacyltransferase.

In a manner similar to that used to deduce the full-length sequence ofDGAT2, a full-length gene encoding the Y. lipolytica PDAT gene wasassembled (2326 bp; SEQ ID NO:45) based on sequencing results.Specifically, the sequence encoded an open reading frame of 1944 bases(nucleotides +274 to +2217 of SEQ ID NO:45), while the deduced aminoacid sequence was 648 residues in length (SEQ ID NO:46). A comparison ofthe deduced amino acid sequence of the Y. lipolytica PDAT (“Yl”) wasmade with other known fungal PDATs (as shown in Table 10) using theanalysis methods described above. TABLE 10 Description of Known orPutative Fungal PDATs Organism Abbreviation Reference Saccharomycescerevisiae Sc Dahlqvist et al., Proc. Natl. Lro 1 gene Acad. Sci. USA97: 6487 (2000) Arabidopsis thaliana At2 GenBank Accession No.“At3g44830” gene NP 190069 [gi: 15230521] (lecithin:cholesterolacyltransferase family protein/LACT family protein) Arabidopsis thalianaAt1 GenBank Accession No. AB006704 [gi: 2351069] Schizosaccharomyces SpGenBank Accession No. pombe “SPBC776.14” NP_596330 gene [gi: 19113122]The results of this comparison are shown as Pair Distances in FIG. 4B.The results demonstrated that the Y. lipolytica PDAT possessed less than47.1% amino acid identity with the other PDAT homologs.

Following sequencing and analysis of the PDAT protein described above,the Yarrowia lipolytica PDAT protein sequence was published as part ofthe Genolevures project (supra). The PDAT sequence disclosed therein wasidentified as ORF YALI-CDS1359.1, encoding 648 amino acids, and theprotein was reported to share some similarities to sp|P40345Saccharomyces cerevisiae YNR008w LRO1, a lecithin cholesterolacyltransferase-like gene which mediates diacylglycerol esterification.

EXAMPLE 7 Functional Expression of Yarrowia lipolytica PDAT inSaccharomvces cerevisiae

The present Example describes the expression of the Yarrowia lipolyticagene (SEQ ID NO:45) encoding PDAT in a wildtype and DGAT2/PDAT knockoutstrain of Saccharomyces cerevisiae.

Saccharomyces cerevisiae Strains

The following two Saccharomyces cerevisiae strains were obtained fromOpen Biosystems (Huntsville, Ala.)

-   -   BY4741 WT (MATa, his3Δ1, leu2Δ0, met15Δ0, and ura3Δ0); and,    -   BY4741 dgal (MATa, his3Δ1, leu2Δ0, met15Δ0, and ura3Δ0), dga1        (comprising a mutant DGAT2 gene).        Haploid strain BY4741 dga1/lro1 was derived from strain BY4741        dga1 by disrupting the Lro1 gene encoding PDAT according to the        methodology recommended by Open Biosytem, as described below.

First, a S. cerevisiae LRO 1 targeting cassette was made by PCRamplifying the S. cerevisiae LEU2 gene from plasmid pJJ250 (Jones, J. S.and I. Prakash, Yeast 6:363-366 (1990)). This was accomplished using thefollowing primer pair:

-   -   UP 161 (SEQ ID NO:84), an 81-mer comprised of 45 bp of 5′        untranslated region of the LRO 1 gene at the primer's 5′ end,        followed by 36 bp of the 5′ end of the LEU2 gene; and,    -   LP 162 (SEQ ID NO:85), an 81-mer comprised of 45 bp of 3′        untranslated region of the LRO 1 gene at the primer's 5′ end,        followed by 36 bp of the 3′ end of the LEU2 gene.

The expected 1901 bp PCR product was purified following agarose gelelectrophoresis and transformed into strain BY4741 dga1 by the standardlithium acetate method (Current Protocols in Molecular Biology, P13.7.1). Transformants were selected on DOB-Leu plates (formulation/L:43.7 g DOBA [BIO 101® Systems, Catalog #4026-012; Krackeler Scientific,Inc., Albany, N.Y.] and 0.69 g CSM-Leu [BIO 101® Systems, Catalog#4510-512; Krackeler Scientific, Inc.]). After 3 days, more than 100transformant colonies were visible; six of these colonies were selectedfor PCR analysis. The LRO 1 knockout was confirmed in all 6 colonies,thus yielding a double knockout of S. cerevisiae, identified herein asstrain BY4741 dga1/lro1.

Synthesis of Plasmid pScGPD-YIPDAT (Comprising a GPD::PDAT::ADH1Chimeric Gene)

The S. cerevisiae GPD (TDH3 gene, encoding glyceraldehyde-3-phosphatedehydrogenase) promoter was amplified using primers GPD-1 (SEQ ID NO:80)and GPD-2 (SEQ ID NO:81), using standard conditions. The 653 bp PCRproduct was cloned into pGEM-T (Promega, Madison, Wis.). The resultingplasmid, pGPD-GEM, was cut with Sac II and Spe I. The 673 bp fragmentcontaining the GPD promoter was isolated and cloned into the S.cerevisiae vector pRS426 digested with Sac II and Spe I, to form plasmidpGPD426 [pRS426 is a yeast autonomously replicating vector that carriesthe URA gene (Christianson T. W., et al., Gene 110:119-122(1992))].

The S. cerevisiae ADH1 (alcohol dehydrogenase gene) terminator regionwas amplified using primers ADHT-1 (SEQ ID NO:82) and ADHT-2 (SEQ IDNO:83). The 330 bp PCR product was cut with Xho I and Kpn I, and clonedinto pGPD426 between Xho I and Kpn I, resulting in formation of plasmidpGPD426N.

Plasmid pGPD426N was cut with Nco I and Not I and then a Nco I-Not Ifragment carrying the Yarrowia PDAT ORF was cloned into it. Thus, theresultant plasmid pScGPD-YIPDAT contained the Yarrowia lipolytica PDATORF under the control of the Saccharomyces cerevisiae GPD promoter(i.e., a GPD::PDAT::ADH1 chimeric gene).

Transformation and Expression of the Yarrowia lipolytica PDAT inSaccharomvces cerevisiae

Saccharomyces cerevisiae strain BY4741 dga1/lro1 was transformed by thestandard lithium acetate method (supra) with either pGPD426N (the“control”) or with yeast plasmid pScGPD-YIPDAT (comprisingGOD::PDAT::ADH1). Positive transformants (i.e., URA prototrophs) werepicked and streaked onto Ura dropout plates (i.e., DOB-Ura plates(formulation/L: 43.7 g DOBA [BIO 101® Systems, Catalog #4026-012;Krackeler Scientific, Inc., Albany, N.Y.] and 0.69 g CSM-Leu [BIO 101®Systems, Catalog #4511-212; Krackeler Scientific, Inc.])) andpre-cultivated for 1-2 days. A loop of cells was picked and inoculatedinto 3 mL Ura dropout medium and cultivated overnight at 30° C. Thepreculture was transferred to 40 mL medium and cells were grown for 52hr prior to being harvested, washed in water, and lyophilized. The drycell weight (“dcw”) was determined and dry cell mass was analyzed bydirect base transesterification. TABLE 11 Lipid Content In Saccharomycescerevisiae Strains Disrupted In PDAT And DGAT2 mg of dcw TFA TFA StrainPlasmid used for GC mg % dcw BY4741 pGPD426N 8.3 67 0.8 dga1/Iro1(control) BY4741 pScGPD- 9.4 154 1.6 dga1/Iro1 YIPDATTotal fatty acids, measured as a percent of the dry cell weight (column5, “TFA % dcw”) was doubled in the pScGPD-YIPDAT transformant ascompared to that in the control (comprising the vector alone). SinceSaccharomyces cerevisiae is not an oleaginous organism, this differencein the amount of total fatty acids produced is significant. Theseresults confirmed that the enzyme encoded by SEQ ID NO:45 corresponds toa functional Yarrowia lipolytica PDAT enzyme.

EXAMPLE 8 Isolation of the Yarrowia Glyceraldehyde PhosphateDehydrogenase (GPD) Promoter Region

The present Example describes the identification of the promoter region(SEQ ID NO:56) of the Yarrowia lipolytica gene encoding glyceraldehydephosphate dehydrogenase, by use of primers derived from conservedregions of other GPD sequences.

A comparison of the various protein sequences encoding GPD genes fromSaccharomyces cerevisiae (GenBank Accession No. CAA24607; SEQ ID NO:57),Schizosaccharomyces pombe (GenBank Accession No. NP_(—)595236; SEQ IDNO:58), Aspergillus oryzae (GenBank Accession No. AAK08065; SEQ IDNO:59), Paralichthys olivaceus (GenBank Accession No. BAA88638; SEQ IDNO:60), Xenopus laevis (GenBank Accession No. P51469; SEQ ID NO:61) andGallus gallus (GenBank Accession No. DECHG3; SEQ ID NO:62) showed thatthere were several stretches of conserved amino acid sequence betweenthe 6 different organisms (FIGS. 5A and 5B). Thus, two degeneratedoligonucleotides (shown below), corresponding to the conserved ‘KYDSTHG’(SEQ ID NO:63) and ‘TGMKAV’ (SEQ ID NO:64) amino acid sequences,respectively, were designed and used to amplify a portion of the codingregion of GPD from Y. lipolytica: Degenerated oligonucleotide YL193:(SEQ ID NO:65) AAGTACGAYTCBACYCAYGG Degenerated oligonucleotide YL194:(SEQ ID NO:66) ACRGCCTTRGCRGCDCCRGT[Note:The nucleic acid degeneracy code used for SEQ ID NOs:65 and 66 was asfollows: R = A/G; Y = C/T; B = C/G/T; and D = A/G/T.]Based on the full-length sequences of the GPD sequences of FIGS. 5A and5B, it was hypothesized that the Yarrowia lipolytica GPD gene amplifiedas described above would be missing ˜50 amino acids from its N-terminusand about ˜115 amino acids from its C-terminus.

The PCR amplification was carried out in a 50 μl total volumecomprising: PCR buffer (containing 10 mM KCl, 10 mM (NH₄)₂SO₄, 20 mMTris-HCl (pH 8.75), 2 mM MgSO₄, 0.1% Triton X-100), 100 μg/mL BSA (finalconcentration), 200 μM each deoxyribonucleotide triphosphate, 10 pmoleof each primer, 50 ng genomic DNA of Y. lipolytica (ATCC #76982) and 1μl of Taq DNA polymerase (Epicentre Technologies). The thermocyclerconditions were set for 35 cycles at 95° C. for 1 min, 56° C. for 30 secand 72° C. for 1 min, followed by a final extension at 72° C. for 10min.

The PCR products were purified using a Qiagen PCR purification kit(Valencia, Calif.), and then further purified following gelelectrophoresis in 1% (w/v) agarose. Subequently, the PCR products werecloned into the pGEM-T-easy vector (Promega, Madison, Wis.). The ligatedDNA was used to transform cells of E. coli DH5α and transformants wereselected on LB agar containing ampicillin (100 μg/mL). Analysis of theplasmid DNA from one transformant confirmed the presence of a plasmid ofthe expected size, which was designated as “pT-GPD”.

Sequence analyses showed that pT-GPD contained a 507 bp fragment (SEQ IDNO:67). Identity of this sequence was determined by conducting BLAST(Basic Local Alignment Search Tool; Altschul, S. F., et al., J. Mol.Biol. 215:403-410 (1993); searches for similarity to sequences containedin the BLAST “nr” database (comprising all non-redundant GenBank CDStranslations, sequences derived from the 3-dimensional structureBrookhaven Protein Data Bank, the SWISS-PROT protein sequence database,EMBL and DDBJ databases). The sequence was analyzed for similarity toall publicly available DNA sequences contained in the “nr” databaseusing the BLASTN algorithm provided by the National Center forBiotechnology Information (NCBI). The DNA sequence was translated in allreading frames and compared for similarity to all publicly availableprotein sequences contained in the “nr” database, using the BLASTXalgorithm (Gish, W. and States, D. J. Nature Genetics 3:266-272 (1993))provided by the NCBI. The results of the BLAST comparison summarizingthe sequence to which SEQ ID NO:67 has the most similarity are reportedaccording to the % identity, % similarity, and Expectation value. “%Identity” is defined as the percentage of amino acids that are identicalbetween the two proteins. “% Similarity” is defined as the percentage ofamino acids that are identical or conserved between the two proteins.“Expectation value” estimates the statistical significance of the match,specifying the number of matches, with a given score, that are expectedin a search of a database of this size absolutely by chance. The 507 bpof pT-GPD was found to encode 169 amino acids (SEQ ID NO:68). This aminoacid fragment had 77% identity and 84% similarity with the GPD proteinsequence of fission yeast (GenBank Accession No. NP_(—)595236), with anexpectation value of 6e-68. The Yarrowia sequence possessed the‘KYDSTHG’ (SEQ ID NO:63) and ‘TGMKAV’ (SEQ ID NO:64) amino acidsequences (corresponding to the degenerate primers used to amplify thefragment) at its N- and C-termini.

To isolate the GPD promoter regions, a genome-walking technique (TOPO®Walker Kit, Invitrogen) was utilized, as described in Example 2.Briefly, genomic DNA of Y. lipolytica was digested with KpnI, SacI, SphIor PacI, and dephosphorylated with Calf Intestinal Alkaline Phosphatase(CIP). Primer extension reactions were then carried out using primerYL206 (SEQ ID NO:69). The primer extended products were linked withTOPO® Linker and then used as template in PCR reactions with LinkAmpPrimer1 (SEQ ID NO:29) and primer YL207 (SEQ ID NO:70). The newlyamplified product was subjected to a second PCR reaction using theLinkAmp primer 2 (SEQ ID NO:77) and YL208 (SEQ ID NO:71) primers.

The PCR products comprising the 5′ upstream region of the GPD gene werepurified using a Qiagen PCR purification kit, followed by gelelectrophoresis in 1% (w/v) agarose. Products were then cloned into thepGEM-T-easy vector (Promega, Madison, Wis.). The ligated DNA was used totransform E. coli DH5α and transformants were selected on LB agarcontaining ampicillin (100 μg/mL).

Analysis of the plasmid DNA from one transformant comprising the 5′upstream region of the GPD gene confirmed the presence of the expectedplasmid, designated pT-GPDP. Sequence analyses showed that pT-GPDPcontained a fragment of 1848 bp (SEQ ID NO:72), which included 1525 bpof 5′ upstream sequence from the nucleotide‘A’ (designated as +1) of thetranslation initiation codon ‘ATG’ of the GPD gene. The nucleotideregion between the −968 position and the ATG translation initiation siteof the GPD gene was determined to contain the putative promoter region(“GPDPro”, provided as SEQ ID NO:56).

EXAMPLE 9 (Prophetic) Expression of Yarrowia lipolytica PDAT and DGAT2ORFs under the Control of a Yarrowia Promoter

The present Example describes the over-expression of the PDAT and DGAT2ORFs in chimeric genes under the control of a Yarrowia lipolyticapromoter in a wild type Yarrowia strain.

Expression of Y. lipolytica DGAT2 in Yarrowia lipolytica

The ORF of Y. lipolytica DGAT2, i.e., SEQ ID NO:86 which encodes theprotein of 355 amino acid residues provided herein as SEQ ID NO:79, wasPCR-amplified using upper primer P145 (SEQ ID NO:73) and lower primerP146 (SEQ ID NO:74) from the genomic DNA of Y. lipolytica ATCC #90812.The expected 1071 bp fragment was isolated, purified, digested with NcoI and Not I and cloned into Nco I-Not I cut pY5-13 vector (described inExample 1), such that the gene was under the control of the Y.lipolytica TEF promoter. Correct transformants were confirmed byminiprep analysis and the resultant plasmid was designated aspY27-DGAT2.

Plasmids pY5-13 (the “control”) and pY27-DGAT2 will be transformed intoY. lipolytica ATCC #90812 wild-type (WT) and DGAT2-disrupted ATCC #90812(“S-D”) strains and selected on BIO 101® Systems DOB/CSM-Leu plates(Krackeler Scientific, Inc., Albany, N.Y.). Single colonies oftransformants will be grown up and GC analyzed, as described in theGeneral Methods.

Expression of Y. lipolytica PDAT in Yarrowia lipolytica

The ORF of Y. lipolytica PDAT was PCR-amplified using primers YPDAT5(SEQ ID NO:75) and YPDAT3 (SEQ ID NO:76) and genomic DNA from Y.lipolytica ATCC #90812 as the template. The expected 1947 bp fragmentwas isolated, purified, digested with Not I and cloned into Not I cutvector pY5-22GPD under the control of the Yarrowia GPD promoter. VectorpY5-22GPD is similar to pY5-13 (Example 1), having an E. coli ApR gene,E. coli ori and Yarrowia ARS sequence. Correct transformants wereconfirmed by analysis of plasmid DNA and the resultant plasmid wasdesignated as pY27-PDAT.

Plasmids pY5-22GPD (the “control”) and pY27-PDAT will be transformedinto Y. lipolytica ATCC #90812 wild-type (WT) and PDAT-disrupted ATCC#90812 (“S-P”) strains and selected on BIO 101® Systems DOB/CSM-Leuplates. Single colonies of transformants will be grown up and GCanalyzed, as described in the General Methods.

Expected Results

Since both PDAT and DGAT2 enzymes are involved in oil biosynthesis,their over-expression is expected to result in increased oil contentunder conditions when these enzymes are limiting. This is supported byresults that demonstrated disruption of DGAT2, PDAT and both genes incombination resulted in lower oil content.

1-16. (canceled)
 17. A method of increasing triacylglycerol content in atransformed host cell comprising: (a) providing a transformed host cellcomprising: (i) at least one gene encoding an acyltransferase enzymehaving the amino acid sequence selected from the group consisting of SEQID NOs:31, 78, 79 and 46 under the control of suitable regulatorysequences; and (ii) a source of fatty acids; (b) growing the cell ofstep (a) under conditions whereby the at least one gene encoding anacyltransferase enzyme is expressed, resulting in the transfer of thefatty acids to triacylglycerol; and (c) optionally recovering thetriacylglycerol of step (b).
 18. A method of increasing the ω-3 or ω-6fatty acid content of triacylglycerols in a transformed host cellcomprising: (a) providing a transformed host cell comprising: (i) atleast one gene encoding at least one enzyme of the ω-3/ω-6 fatty acidbiosynthetic pathway; (ii) at least one gene encoding an acyltransferaseenzyme having the amino acid sequence selected from the group consistingof SEQ ID NOs:31, 78, 79 and 46 under the control of suitable regulatorysequences; (b) growing the cell of step (a) under conditions whereby thegenes of (i) and (ii) are expressed, resulting in the production of atleast one ω-3 or ω-6 fatty acid and its transfer to triacylglycerol; and(c) optionally recovering the triacylglycerol of step (b).
 19. A methodaccording to claim 18, wherein the at least one gene encoding at leastone enzyme of the ω-3/ω-6 fatty acid biosynthetic pathway is selectedfrom the group consisting of desaturases and elongases.
 20. A methodaccording to claim 19, wherein the desaturase is selected from the groupconsisting of: Δ9 desaturase, Δ12 desaturase, Δ6 desaturase, Δ5desaturase, Δ17 desaturase, a Δ8 desaturase, Δ15 desaturase and Δ4desaturase.
 21. A method according to any of claims 17 or 18, whereinthe any of claims 17 or 18, wherein the host cell is selected from thegroup consisting of algae, bacteria, molds, fungi and yeasts.
 22. Amethod according to claim 21, wherein the host cell is an oleaginousyeast.
 23. A method according to claim 22 wherein the oleaginous yeastis a member of a genus selected from the group of consisting ofYarrowia, Candida, Rhodotorula, Rhodosporidium, Cryptococcus,Trichosporon and Lipomyces.
 24. A method according to claim 23, whereinthe oleaginous yeast is Yarrowia lipolytica.
 25. A method according toclaim 24, wherein the Yarrowia lipolytica is a strain selected from thegroup consisting of Yarrowia lipolytica ATCC #20362, Yarrowia lipolyticaATCC #8862, Yarrowia lipolytica ATCC #18944, Yarrowia lipolytica ATCC#76982, Yarrowia lipolytica ATCC #90812 and Yarrowia lipolytica LGAMS(7)1.
 26. A method according to claim 17 wherein the fatty acid isselected from the group consisting of: stearate, oleic acid, linoleicacid, γ-linoleic acid, dihomo- γ-linoleic acid, arachidonic acid,α-linoleic acid, steraidonic acid, eicosatetraenoic acid,eicosapentaenoic acid docosapentaenoic acid, eicosadienoic acid andeicosatrienoic acid.